New Pyridyl and Dihydroisoquinoline Alkaloids Isolated from the Chevron Nemertean Amphiporus angulatus.

Nemertean worms contain toxins that are used to paralyze their prey and to deter potential predators. Hoplonemerteans often contain pyridyl alkaloids like anabaseine that act through nicotinic acetylcholine receptors and crustacean chemoreceptors. The chemical reactivity of anabaseine, the first nemertean alkaloid to be identified, has been exploited to make drug candidates selective for alpha7 subtype nAChRs. GTS-21, a drug candidate based on the anabaseine scaffold, has pro-cognitive and anti-inflammatory actions in animal models. The circumpolar chevron hoplonemertean Amphiporus angulatus contains a multitude of pyridyl compounds with neurotoxic, anti-feeding, and anti-fouling activities. Here, we report the isolation and structural identification of five new compounds, doubling the number of pyridyl alkaloids known to occur in this species. One compound is an isomer of the tobacco alkaloid anatabine, another is a unique dihydroisoquinoline, and three are analogs of the tetrapyridyl nemertelline. The structural characteristics of these ten compounds suggest several possible pathways for their biosynthesis.

Discovery of the Nicotinic Receptor Toxin Anabaseine in a Polystiliferan Nemertean.

Nemerteans (also called Nemertines) are a phylum of predominantly marine worms that use toxins to capture prey and to defend themselves against predators. Hoplonemerteans have a proboscis armed with one or more stylets used in prey capture and are taxonomically divided into Order Monostilifera, whose members possess a single large proboscis stylet, and Order Polystilifera, whose members have multiple small stylets. Many monostiliferans contain alkaloidal toxins, including anabaseine, that stimulate and then desensitize nicotinic acetylcholine receptors that are present in all animals. These compounds also interact with pyridyl chemoreceptors in crustaceans, reducing predation and larval settlement. Anabaseine has been a lead compound in the design of alpha7 nicotinic acetylcholine receptor agonists like GTS-21 (also called DMXBA) to treat disorders of cognition such as Alzheimer's disease and schizophrenia. These drug candidates also display anti-inflammatory activities of potential medical importance. Most polystiliferans live deep in open oceans and are relatively inaccessible. We fortunately obtained two live specimens of a large benthic polystiliferan, Paradrepanophorus crassus (Pc), from the coast of Spain. MS and NMR analyses of the Ehrlich's reagent derivative allowed identification of anabaseine. A spectrophotometric assay for anabaseine, also based on its reaction with Ehrlich's reagent, revealed high concentrations of anabaseine in the body and proboscis. Apparently, the biosynthetic mechanism for producing anabaseine was acquired early in the evolution of the Hoplonemertea, before the monostiliferan-polystiliferan divergence.

The nonopioid cholinergic agonist GTS-21 mitigates morphine-induced aggravation of burn injury pain together with inhibition of spinal microglia activation in young rats.

BACKGROUND: Repetitive opioid use does not always alleviate basal pain, procedural pain, or both after burn injury. Mitigation of burn injury-site pain can be achieved by GTS-21 stimulation of α7-acetylcholine nicotinic receptors (α7AChRs) and reduced microglia activation in rat. We tested the hypothesis that morphine exaggerates burn injury-site pain and GTS-21 alleviates both morphine-induced aggravated burn injury pain and microglia activation. METHODS: Young rats with dorsal paw burn injury or sham-burn received intraperitoneal saline, morphine, GTS-21, or a combination twice daily for 14 days. Ipsilateral plantar pain thresholds were tested every other day before morning drugs from days 0-20. Spinal microglia activation, evidenced as pain-transducer (tumour necrosis factor-α [TNF-α], interleukin [IL]-6, IL-1ß, nuclear factor kappa B [NF-κB], Toll-like receptor 4 [TLR4]) expression, was examined using immunohistochemistry and immunoblot. In cultured microglia, morphine-induced cytokine expression was measured (quantitative polymerase chain reaction/enzyme-linked immunosorbent assay [qPCR/ELISA]). RESULTS: Morphine aggravated allodynia at day 5 in sham-burn (P=0.039, n=8-11) but significantly aggravated burn injury site allodynia by day 3 (P=0.010, n=8-11). Microgliosis paralleled nociceptive behaviour changes where burn injury with morphine had highest microgliosis compared with burn injury, morphine alone, or controls (number of cells per field [SD]: 33.8 [2.4], 18.0 [4.1], 8.2 [1.9], and 4.8 [2.0], respectively; P<0.001, n=4-5]. GTS-21 reversed the morphine-induced pain component in sham-burn and burn injury rats together with reduced microgliosis and spinal pain-transducer expression (TNF-α, IL-6, IL-1ß, NF-κB, and TLR4). Morphine-exposed microglial cells showed increased cytokine expression, which was mitigated by GTS-21. CONCLUSIONS: Morphine or burn injury alone increases pain together with microgliosis and pain-transducer expression. Morphine administration augments burn injury-site nociception sooner and aggravated spinal microgliosis and inflammatory pain-transducer expression. GTS-21 has the potential to treat morphine-induced pain in burn injury.

Interactions of Nereistoxin and Its Analogs with Vertebrate Nicotinic Acetylcholine Receptors and Molluscan ACh Binding Proteins.

Nereistoxin (NTX) is a marine toxin isolated from an annelid worm that lives along the coasts of Japan. Its insecticidal properties were discovered decades ago and this stimulated the development of a variety of insecticides such as Cartap that are readily transformed into NTX. One unusual feature of NTX is that it is a small cyclic molecule that contains a disulfide bond. In spite of its size, it acts as an antagonist at insect and mammalian nicotinic acetylcholine receptors (nAChRs). The functional importance of the disulfide bond was assessed by determining the effects of inserting a methylene group between the two sulfur atoms, creating dimethylaminodithiane (DMA-DT). We also assessed the effect of methylating the NTX and DMA-DT dimethylamino groups on binding to three vertebrate nAChRs. Radioligand receptor binding experiments were carried out using washed membranes from rat brain and fish (Torpedo) electric organ; [3H]-cytisine displacement was used to assess binding to the predominantly high affinity alpha4beta2 nAChRs and [125I]-alpha-bungarotoxin displacement was used to measure binding of NTX and analogs to the alpha7 and skeletal muscle type nAChRs. While the two quaternary nitrogen analogs, relative to their respective tertiary amines, displayed lower α4ß2 nAChR binding affinities, both displayed much higher affinities for the Torpedo muscle nAChR and rat alpha7 brain receptors than their respective tertiary amine forms. The binding affinities of DMA-DT for the three nAChRs were lower than those of NTX and MeNTX. An AChBP mutant lacking the C loop disulfide bond that would potentially react with the NTX disulfide bond displayed an NTX affinity very similar to the parent AChBP. Inhibition of [3H]-epibatidine binding to the AChBPs was not affected by exposure to NTX or MeNTX for up to 24 hr prior to addition of the radioligand. Thus, the disulfide bond of NTX is not required to react with the vicinal disulfide in the AChBP C loop for inhibition of [3H]-epibatidine binding. However, a reversible disulfide interchange reaction of NTX with nAChRs might still occur, especially under reducing conditions. Labeled MeNTX, because it can be readily prepared with high specific radioactivity and possesses relatively high affinity for the nAChR-rich Torpedo nAChR, would be a useful probe to detect and identify any nereistoxin adducts.

Systemic Administration of α7-Nicotinic Acetylcholine Receptor Ligands Does Not Improve Renal Injury or Behavior in Mice With Advanced Systemic Lupus Erythematosus.

There is a critical need for safe treatment options to control inflammation in patients with systemic lupus erythematosus (SLE) since the inflammation contributes to morbidity and mortality in advanced disease. Endogenous neuroimmune mechanisms like the cholinergic anti-inflammatory pathway can be targeted to modulate inflammation, but the ability to manipulate such pathways and reduce inflammation and end organ damage has not been fully explored in SLE. Positive allosteric modulators (PAM) are pharmacological agents that inhibit desensitization of the nicotinic acetylcholine receptor (α7-nAChR), the main anti-inflammatory feature within the cholinergic anti-inflammatory pathway, and may augment α7-dependent cholinergic tone to generate therapeutic benefits in SLE. In the current study, we hypothesize that activating the cholinergic anti-inflammatory pathway at the level of the α7-nAChR with systemic administration of a partial agonist, GTS-21, and a PAM, PNU-120596, would reduce inflammation, eliminating the associated end organ damage in a mouse model of SLE with advanced disease. Further, we hypothesize that systemic α7 ligands will have central effects and improve behavioral deficits in SLE mice. Female control (NZW) and SLE mice (NZBWF1) were administered GTS-21 or PNU-120596 subcutaneously via minipumps for 2 weeks. We found that the increased plasma dsDNA autoantibodies, splenic and renal inflammation, renal injury and hypertension usually observed in SLE mice with advanced disease at 35 weeks of age were not altered by GTS-21 or PNU-120596. The anxiety-like behavior presented in SLE mice was also not improved by GTS-21 or PNU-120596. Although no significant beneficial effects of α7 ligands were observed in SLE mice at this advanced stage, we predict that targeting this receptor earlier in the pathogenesis of the disease may prove to be efficacious and should be addressed in future studies.

Therapeutic efficacy of α7 ligands after acute ischaemic stroke is linked to conductive states of α7 nicotinic ACh receptors.

BACKGROUND AND PURPOSE: Targeting α7 nicotinic ACh receptors (nAChRs) in neuroinflammatory disorders including acute ischaemic stroke holds significant therapeutic promise. However, therapeutically relevant signalling mechanisms remain unidentified. Activation of neuronal α7 nAChRs triggers ionotropic signalling, but there is limited evidence for it in immunoglial tissues. The α7 ligands which are effective in reducing acute ischaemic stroke damage promote α7 ionotropic activity, suggesting a link between their therapeutic effects for treating acute ischaemic stroke and activation of α7 conductive states. EXPERIMENTAL APPROACH: This hypothesis was tested using a transient middle cerebral artery occlusion (MCAO) model of acute ischaemic stroke, NS6740, a known selective non-ionotropic agonist of α7 nAChRs and 4OH-GTS-21, a partial α7 agonist. NS6740-like ligands exhibiting low efficacy/potency for ionotropic activity will be referred to as non-ionotropic agonists or "metagonists". KEY RESULTS: 4OH-GTS-21, used as a positive control, significantly reduced neurological deficits and brain injury after MCAO as compared to vehicle and NS6740. By contrast, NS6740 was ineffective in identical assays and reversed the effects of 4OH-GTS-21 when these compounds were co-applied. Electrophysiological recordings from acute hippocampal slices obtained from NS6740-injected animals demonstrated its remarkable brain availability and protracted effects on α7 nAChRs as evidenced by sustained (>8 h) alterations in α7 ionotropic responsiveness. CONCLUSION AND IMPLICATIONS: These results suggest that α7 ionotropic activity may be obligatory for therapeutic efficacy of α7 ligands after acute ischaemic stroke yet, highlight the potential for selective application of α7 ligands to disease states based on their mode of receptor activation.

Nonopioid GTS-21 Mitigates Burn Injury Pain in Rats by Decreasing Spinal Cord Inflammatory Responses.

BACKGROUND: Burn injury (BI) pain consists of inflammatory and neuropathic components and activates microglia. Nicotinic alpha 7 acetylcholine receptors (α7AChRs) expressed in microglia exhibit immunomodulatory activity during agonist stimulation. Efficacy of selective α7AChR agonist GTS-21 to mitigate BI pain and spinal pain-mediators was tested. METHODS: Anesthetized rats after hind-paw BI received intraperitoneal GTS-21 or saline daily. Allodynia and hyperalgesia were tested on BI and contralateral paw for 21 days. Another group after BI receiving GTS-21 or saline had lumbar spinal cord segments harvested (day 7 or 14) to quantify spinal inflammatory-pain transducers or microglia activation using fluorescent marker, ionized calcium-binding adaptor protein (Iba1). RESULTS: BI significantly decreased allodynia withdrawal threshold from baseline of ~9-10 to ~0.5-1 g, and hyperalgesia latency from ~16-17 to ~5-6 seconds by day 1. Both doses of GTS-21 (4 or 8 mg/kg) mitigated burn-induced allodynia from ~0.5-1 to ~2-3 g threshold (P = .089 and P = .010), and hyperalgesia from ~5-6 to 8-9 seconds (P < .001 and P < .001) by day 1. The GTS-21 group recovered to baseline pain threshold by day 15-17 compared to saline-treated, where the exaggerated nociception persisted beyond 15-17 days. BI significantly (P < .01) increased spinal cord microgliosis (identified by fluorescent Iba1 staining), microglia activation (evidenced by the increased inflammatory cytokine), and pain-transducer (protein and/or messenger RNA [mRNA]) expression (tumor necrosis factor-α [TNF-α], interleukin-1ß [IL-1ß], nuclear factor-kappa B [NF-κB], interleukin-6 [IL-6], Janus-associated kinase signal transducer and activator of transcription 3 [JAK-STAT3], and/or N-methyl-D-aspartate receptor [NMDAR]). GTS-21 mitigated pain-transducer changes. The α7AChR antagonist methyllycaconitine nullified the beneficial effects of GTS-21 on both increased nociception and pain-biomarker expression. CONCLUSIONS: Nonopioid, α7AChR agonist GTS-21 elicits antinociceptive effects at least in part by decreased activation spinal-cord pain-inducers. The α7AChR agonist GTS-21 holds promise as potential therapeutic adjunct to decrease BI pain by attenuating both microglia changes and expression of exaggerated pain transducers.

Spleen is not required for therapeutic effects of 4OH-GTS-21, a selective α7 nAChR agonist, in the sub-acute phase of ischemic stroke in rats.

Acute ischemic stroke (AIS) causes both central and peripheral inflammation, while activation of α7 nicotinic acetylcholine receptors (nAChRs) provides both central and peripheral anti-inflammatory and anti-apoptotic effects. Here, we provide evidence that 4OH-GTS-21, a selective α7 agonist, produces its therapeutic effects via primarily central sites of action because 4OH-GTS-21 was found equally effective in splenectomized and non-spenectomized rats in the sub-acute phase of ischemic stroke (≤1 week). However, the spleen may boost the therapeutic efficacy of 4OH-GTS-21 in certain behavioral tasks as our data also indicated. In our tests, AIS was modeled by transient middle cerebral artery occlusion (tMCAO). Splenectomy was done 2 weeks before tMCAO. We determined that: 1) Daily 4OH-GTS-21 treatments for 7 days after tMCAO significantly reduced neurological deficits and brain injury in both splenectomized and non-spelenectomized rats demonstrating that the spleen is not required for therapeutic benefits of 4OH-GTS-21; 2) The effects of 4OH-GTS-21 in the adhesive sticker removal test were significantly weaker in splenectomized animals suggesting that the spleen boosts the efficacy of 4OH-GTS-21 in the first week after tMCAO; and 3) Ischemic brain injury was not significantly affected by splenectomy in both vehicle-treated and 4OH-GTS-21-treated animals. These data support the hypothesis that the therapeutic efficacy of sub-chronic (≤1 week) 4OH-GTS-21 primarily originates from central sites of action. These results validate brain availability as a critical factor for developing novel α7 ligands for AIS.

A Methyl Scan of the Pyrrolidinium Ring of Nicotine Reveals Significant Differences in Its Interactions with α7 and α4ß2 Nicotinic Acetylcholine Receptors.

The two major nicotinic acetylcholine receptors (nAChRs) in the brain are the α4ß2 and α7 subtypes. A "methyl scan" of the pyrrolidinium ring was used to detect differences in nicotine's interactions with these two receptors. Each methylnicotine was investigated using voltage-clamp and radioligand binding techniques. Methylation at each ring carbon elicited unique changes in nicotine's receptor interactions. Replacing the 1'-N-methyl with an ethyl group or adding a second 1'-N-methyl group significantly reduced interaction with α4ß2 but not α7 receptors. The 2'-methylation uniquely enhanced binding and agonist potency at α7 receptors. Although 3'- and 5'-trans-methylations were much better tolerated by α7 receptors than α4ß2 receptors, 4'-methylation decreased potency and efficacy at α7 receptors much more than at α4ß2 receptors. Whereas cis-5'-methylnicotine lacked agonist activity and displayed a low affinity at both receptors, trans-5'-methylnicotine retained considerable α7 receptor activity. Differences between the two 5'-methylated analogs of the potent pyridyl oxymethylene-bridged nicotine analog A84543 were consistent with what was found for the 5'-methylnicotines. Computer docking of the methylnicotines to the Lymnaea acetylcholine binding protein crystal structure containing two persistent waters predicted most of the changes in receptor affinity that were observed with methylation, particularly the lower affinities of the cis-methylnicotines. The much smaller effects of 1'-, 3'-, and 5'-methylations and the greater effects of 2'- and 4'-methylations on nicotine α7 nAChR interaction might be exploited for the design of new drugs based on the nicotine scaffold. SIGNIFICANCE STATEMENT: Using a comprehensive "methyl scan" approach, we show that the orthosteric binding sites for acetylcholine and nicotine in the two major brain nicotinic acetylcholine receptors interact differently with the pyrrolidinium ring of nicotine, and we suggest reasons for the higher affinity of nicotine for the heteromeric receptor. Potential sites for nicotine structure modification were identified that may be useful in the design of new drugs targeting these receptors.

A Pharmacological Comparison of Two Isomeric Nicotinic Receptor Agonists: The Marine Toxin Isoanatabine and the Tobacco Alkaloid Anatabine.

Many organisms possess "secondary" compounds to avoid consumption or to immobilize prey. While the most abundant or active compounds are initially investigated, more extensive analyses reveal other "minor" compounds with distinctive properties that may also be of biomedical and pharmaceutical significance. Here, we present an initial in vitro investigation of the actions of two isomeric tetrahydropyridyl ring-containing anabasine analogs: isoanatabine, an alkaloid isolated from a marine worm, and anatabine, a relatively abundant minor alkaloid in commercial tobacco plants. Both compounds have a double bond that is distal to the piperidine ring nitrogen of anabasine. Racemic isoanatabine and anatabine were synthesized and their S- and R-enantiomers were isolated by chiral high pressure liquid chromatography (HPLC). Both isoanatabines displayed higher efficacies at α4ß2 nicotinic acetylcholine receptors (nAChRs) relative to the anatabines; R-isoanatabine was most potent. Radioligand binding experiments revealed similar α4ß2 nAChR binding affinities for the isoanatabines, but R-anatabine affinity was twice that of S-anatabine. While the two anatabines and S-isoanatabine were highly efficacious agonists at α7 nAChRs, R-isoanatabine was only a weak partial agonist. The four compounds share an ability to stimulate both α4ß2 and α7 nAChRs, a property that may be useful in developing more efficacious drugs to treat neurodegenerative and other medical disorders.

GTS-21 attenuates loss of body mass, muscle mass, and function in rats having systemic inflammation with and without disuse atrophy.

Muscle changes of critical illness are attributed to systemic inflammatory responses and disuse atrophy. GTS-21 (3-(2,4-dimethoxy-benzylidene)anabaseine), also known as DMBX-A) is a synthetic derivative of the natural product anabaseine that acts as an agonist at α7-acetylcholine receptors (α7nAChRs). Hypothesis tested was that modulation of inflammation by agonist GTS-21 (10 mg/kg b.i.d. intraperitoneally) will attenuate body weight (BW) and muscle changes. Systemic sham inflammation was produced in 125 rats by Cornyebacterium parvum (C.p.) or saline injection on days 0/4/8. Seventy-four rats had one immobilized-limb producing disuse atrophy. GTS-21 effects on BW, tibialis muscle mass (TMM), and function were assessed on day 12. Systemically, methemoglobin levels increased 26-fold with C.p. (p < 0.001) and decreased significantly (p < 0.033) with GTS-21. Control BW increased (+ 30 ± 9 g, mean ± SD) at day 12, but decreased with C.p. and superimposed disuse (p = 0.005). GTS-21 attenuated BW loss in C.p. (p = 0.005). Compared to controls, TMM decreased with C.p. (0.43 ± 0.06 g to 0.26 ± 0.03 g) and with superimposed disuse (0.18 ± 0.04 g); GTS-21 ameliorated TMM loss to 0.32 ± 0.04 (no disuse, p = 0.028) and to 0.22 ± 0.03 (with disuse, p = 0.004). Tetanic tensions decreased with C.p. or disuse and GTS-21 attenuated tension decrease in animals with disuse (p = 0.006) and in animals with C.p. and disuse (p = 0.029). C.p.-induced 11-fold increased muscle α7nAChR expression was decreased by > 60% with GTS-21 treatment. In conclusion, GTS-21 modulates systemic inflammation, evidenced by both decreased methemoglobin levels and decrease of α7nAChR expression, and mitigates inflammation-mediated loss of BW, TMM, fiber size, and function.

Pharmacokinetic Limitations on Effects of an Alpha7-Nicotinic Receptor Agonist in Schizophrenia: Randomized Trial with an Extended-Release Formulation.

The aim of the trial was to assess whether extending plasma levels of the alpha7-nicotinic acetylcholine receptor (nAChR) agonist 3-(2,4-dimethoxybenzylidene)-anabaseine (DMXB-A) over time enhances its cognitive effects in schizophrenia. Both smoking and non-smoking patients were studied, to determine whether effects differ between these two groups. Forty-three smokers and thirty-seven non-smokers who met DSM-IV criteria for schizophrenia were enrolled in a double-blind, randomized, placebo-controlled 1 month trial. DMXB-A 150 mg was formulated with hypromellose to produce extended release over 4 h and administered four times daily. The primary outcome (the Neurocognitive Composite of the MATRICS Consensus Cognitive Battery) and secondary outcomes (the MATRICS Attention-Vigilance Domain and P50 gating), showed no significant effect. Plasma levels were obtained 2.5 h post administration. In non-smokers, levels were similar to those reached transiently with 75-150 mg DMXB-A immediate-release formulations twice daily, which were earlier shown to be effective doses. However, the extended-release formulation produced no cognitive or clinical effect either in non-smokers or smokers. The 10-fold lower DMXB-A plasma levels in smokers suggest that chronic smoking enhances DMXB-A metabolism. Pro-cognitive effects of DMXB-A may result from transient increases in cell signaling that are limited by receptor tachyphylaxis. Future efforts to improve cognition in schizophrenia by enhancing alpha7 nAChR function may require consideration of these pharmacokinetic limitations.

Alpha7 nicotinic acetylcholine receptor-specific agonist DMXBA (GTS-21) attenuates Aß accumulation through suppression of neuronal γ-secretase activity and promotion of microglial amyloid-ß phagocytosis and ameliorates cognitive impairment in a mouse model of Alzheimer's disease.

We previously demonstrated that stimulation of nicotinic acetylcholine receptors (nAChRs) increases amyloid-ß (Aß) phagocytosis in rat microglia and is closely associated with the decrease of brain Aß and amelioration of memory dysfunction in a transgenic mouse model of Alzheimer's disease (AD). Here, we examined the subtypes of nAChRs involved in these beneficial effects. In primary cultures of rat microglia, the α7 nAChR selective agonist 3-[(2,4-dimethoxy)benzylidene]-anabaseine dihydrochloride (DMXBA) promoted Aß and fluorescent latex bead phagocytosis, whereas selective α7 nAChR antagonists suppressed the enhanced Aß phagocytosis. In a transgenic mouse model of AD, administration of DMXBA attenuated brain Aß burden and memory dysfunction. Moreover, DMXBA suppressed γ-secretase activity in solubilized fractions of human neuroblastoma cells and transgenic mouse brain. These results suggested that selective activation of α7 nAChRs promoted microglial Aß phagocytosis and suppressed neuronal γ-secretase activity to contribute to the attenuation of the brain Aß burden and cognitive impairment. Thus, we propose neuronal and microglial α7 nAChRs as new therapeutic targets in the treatment of AD.

An ALPHA7 Nicotinic Acetylcholine Receptor Agonist (GTS-21) Promotes C2C12 Myonuclear Accretion in Association with Release of Interleukin-6 (IL-6) and Improves Survival in Burned Mice.

The role of interleukin-6 (IL-6) in physiological processes and disease is poorly understood. The hypothesis tested in this study was that selective alpha7 acetylcholine receptor (α7AChR) agonist, GTS-21, releases IL-6 in association with myonuclear accretion and enhances insulin signaling in muscle cells, and improves survival of burn injured (BI) mice. The in vitro effects of GTS-21 were determined in C2C12 myoblasts and 7-day differentiated myotubes (myotubes). The in vivo effects of GTS-21 were tested in BI wild-type (WT) and IL-6 knockout (IL6KO) mice. GTS-21 dose-dependently (0 µM, 100 µM, and 200 µM) significantly increased IL-6 levels in myoblasts and myotubes at 6 and 9 h. GTS-21-induced IL-6 release in myotubes was attenuated by methyllycaconitine (α7AChR antagonist), and by Stat-3 or Stat-5 inhibitors. GTS-21 increased MyoD and Pax7 protein expression, myonuclear accretion, and insulin-induced phosphorylation of Akt, GSK-3ß, and Glut4 in myotubes. The glucose levels of burned IL6KO mice receiving GTS-21 decreased significantly compared with sham-burn IL6KO mice. Superimposition of BI on IL6KO mice caused 100% mortality; GTS-21 reduced mortality to 75% in the IL6KO mice. The 75% mortality in burned WT mice was reduced to 0% with GTS-21. The in vitro findings suggest that GTS-21-induced IL-6 release from muscle is mediated via α7AChRs upstream of Stat-3 and -5 pathways and is associated with myonuclear accretion, possibly via MyoD and Pax7 expression. GTS-21 in vivo improves survival in burned WT mice and IL6KO mice, suggesting a potential therapeutic application of α7AChR agonists in the treatment of BI.

Prevention of Burn-Induced Inflammatory Responses and Muscle Wasting by GTS-21, a Specific Agonist for α7 Nicotinic Acetylcholine Receptors.

INTRODUCTION: Muscle wasting (MW) in catabolic conditions (e.g., burn injury [BI]) is a major risk factor affecting prognosis. Activation of interleukin-1ß (IL-1ß)/nuclear factor-kappa B (NF-κB), interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3), and/or forkhead box O transcriptional factor (FOXO)-mediated gene transcription pathways is the pivotal trigger of inflammatory response-induced protein catabolic processes in muscle. The α7 acetylcholine receptors (α7AChRs) are upregulated in macrophages and peripheral tissues including skeletal muscle during MW conditions. Stimulation of α7AChRs mitigates inflammatory responses. Hypothesis tested is that attenuation of inflammation by α7AChR stimulation with specific α7AChR agonist, GTS-21, will reverse BI-induced body mass and MW by modulating inflammatory and proteolytic signals. METHODS: Body surface area (30%) BI or sham BI mice were treated with GTS-21 or saline. Tibialis anterior (TA) muscle was harvested at 6 h, day 1 or 3 to examine inflammatory and proteolytic signals. RESULTS: GTS-21 significantly ameliorated the BI-induced increased expression of inflammatory cytokines IL-6, IL-1ß, C-X-C motif chemokine ligand 2 (6 h), phosphorylated STAT3, and NF-κB (day 1) in TA muscle. GTS-21 also significantly inhibited BI-induced increase of MuRF1 and FOXO1 (day 1). Consistent with the cytokine and inflammatory mediator changes, BI-induced body weight and TA muscle mass loss at day 3 were mitigated by GTS-21 treatment. The beneficial effect of GTS-21 on BI changes was absent in methyllycaconitine (α7AChR antagonist)-treated wild-type and α7AChR knockout mice. CONCLUSION: GTS-21 stimulation of α7AChRs, by modulating multiple molecular signals related to inflammation and proteolysis, attenuates protein wasting, evidenced by maintenance of body weight and attenuation of distant muscle mass loss after BI. GTS-21 can be a novel, potent therapeutic option for reversal of BI-induced MW.

Brief Report: Initial Trial of Alpha7-Nicotinic Receptor Stimulation in Two Adult Patients with Autism Spectrum Disorder.

Abnormalities in CHRNA7, the alpha7-nicotinic receptor gene, have been reported in autism spectrum disorder. These genetic abnormalities potentially decrease the receptor's expression and diminish its functional role. This double-blind, placebo-controlled crossover study in two adult patients investigated whether an investigational receptor-specific partial agonist drug would increase the inhibitory functions of the gene and thereby increase patients' attention. An electrophysiological biomarker, P50 inhibition, verified the intended neurobiological effect of the agonist, and neuropsychological testing verified a primary cognitive effect. Both patients perceived increased attention in their self-ratings. Alpha7-nicotinic receptor agonists, currently the target of drug development in schizophrenia and Alzheimer Disease, may also have positive clinical effects in autism spectrum disorder.

Activation and Desensitization of Peripheral Muscle and Neuronal Nicotinic Acetylcholine Receptors by Selected, Naturally-Occurring Pyridine Alkaloids.

Teratogenic alkaloids can cause developmental defects due to the inhibition of fetal movement that results from desensitization of fetal muscle-type nicotinic acetylcholine receptors (nAChRs). We investigated the ability of two known teratogens, the piperidinyl-pyridine anabasine and its 1,2-dehydropiperidinyl analog anabaseine, to activate and desensitize peripheral nAChRs expressed in TE-671 and SH-SY5Y cells. Activation-concentration response curves for each alkaloid were obtained in the same multi-well plate. To measure rapid desensitization, cells were first exposed to five potentially-desensitizing concentrations of each alkaloid in log10 molar increments from 10 nM to 100 µM and then to a fixed concentration of acetylcholine (ACh), which alone produces near-maximal activation. The fifty percent desensitization concentration (DC50) was calculated from the alkaloid concentration-ACh response curve. Agonist fast desensitization potency was predicted by the agonist potency measured in the initial response. Anabaseine was a more potent desensitizer than anabasine. Relative to anabaseine, nicotine was more potent to autonomic nAChRs, but less potent to the fetal neuromuscular nAChRs. Our experiments have demonstrated that anabaseine is more effective at desensitizing fetal muscle-type nAChRs than anabasine or nicotine and, thus, it is predicted to be more teratogenic.

Recombinant expression and predicted structure of parborlysin, a cytolytic protein from the Antarctic heteronemertine Parborlasia corrugatus.

The heteronemertine Parborlasia corrugatus contains a cytolytic protein, parborlysin, which after extensive purification was found by Edman sequencing to be a mixture of several homologues. To investigate this microheterogeneity and enable the analysis of single toxins, we have obtained seven parborlysin isoform genes from P. corrugatus collected in Antarctica. Total RNA was isolated from the homogenized head region and parborlysin genes were identified from a cDNA library using degenerate primers. The translated sequences reveal that the isoforms are ∼ 10 kDa basic (pI ∼ 10) proteins of which all but one harbour six cysteine residues. We generated a model of the three dimensional structure of parborlysins, which suggests that they are composed of five alpha-helical segments that include large, exposed hydrophobic surfaces. Finally, we constructed plasmids and inserted them into Escherichia coli to obtain overexpressed amino- or carboxy-terminal polyhistidine-tagged parborlysin isoforms fused to the third domain of the E. coli periplasmic-protein TolA to facilitate toxin isolation. One of the isoforms adversely affected growth in the E. coli expressing it. Although we succeeded in isolating one of the recombinant parborlysin constructs, it lacked haemolytic activity.