Dietary alkaloid sequestration in a poison frog:\ud an experimental test of alkaloid uptake in Melanophryniscus\ud stelzneri (Bufonidae)

Several lineages of brightly colored anurans independently evolved the ability to secrete alkaloid-containing defensive chemicals from granular glands in the skin. These species, collectively referred to as ‘poison frogs,’ form a polyphyletic assemblage that includes some species of Dendrobatidae, Mantellidae, Myobatrachidae, Bufonidae, and Eleutherodactylidae. The ability to sequester alkaloids from dietary arthropods has been demonstrated experimentally in most poison frog lineages but not in bufonid or eleutherodactylid poison frogs. As with other poison frogs, species of the genus Melanophryniscus (Bufonidae) consume large numbers of mites and ants, suggesting they might also sequester defensive alkaloids from dietary sources. To test this hypothesis, fruit flies dusted with alkaloid/nutritional supplement powder were fed to individual Melanophryniscus stelzneri in two experiments. In the first experiment, the alkaloids 5,8-disubstituted indolizidine 235B' and decahydroquinoline were administered to three individuals for 104 days. In the second experiment, the alkaloids 3,5-disubstituted indolizidine 239Q and decahydroquinoline were given to three frogs for 153 days. Control frogs were fed fruit flies dusted only with nutritional supplement. Gas chromatography/mass spectrometry analyses revealed that skin secretions of all experimental frogs contained alkaloids, whereas those of all control frogs lacked alkaloids. Uptake of decahydroquinoline was greater than uptake of 5,8-disubstituted indolizidine, and uptake of 3,5-disubstituted indolizidine was greater than uptake of decahydroquinoline, suggesting greater uptake efficiency of certain alkaloids. Frogs in the second experiment accumulated a greater amount of alkaloid, which corresponds to the longer duration and greater number of alkaloid-dusted fruit flies that were consumed. These findings provide the first experimental evidence that bufonid poison frogs sequester alkaloid-based defenses from dietary sourcesWe thank V. Korb for her assistance in frog feeding, and M.A. Nichols for invaluable assistance in maintaining the GC/MS instrument. We extend a special thanks to C.D. Anthony, C. Hickerson, and C.A. Sheil for providing suggestions and comments that improved the quality of this manuscript.TG was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico Proc. 307001/ 2011-3 and Fundação de Amparo à Pesquisa do Estado de São Paulo Proc. 2012/10000-5

Speculation on How RIC-3 and Other Chaperones Facilitate α7 Nicotinic Receptor Folding and Assembly

The process of how multimeric transmembrane proteins fold and assemble in the endoplasmic reticulum is not well understood. The alpha7 nicotinic receptor (α7 nAChR) is a good model for multimeric protein assembly since it has at least two independent and specialized chaperones: Resistance to Inhibitors of Cholinesterase 3 (RIC-3) and Nicotinic Acetylcholine Receptor Regulator (NACHO). Recent cryo-EM and NMR data revealed structural features of α7 nAChRs. A ser-ala-pro (SAP) motif precedes a structurally important but unique “latch” helix in α7 nAChRs. A sampling of α7 sequences suggests the SAP motif is conserved from C. elegans to humans, but the latch sequence is only conserved in vertebrates. How RIC-3 and NACHO facilitate receptor subunits folding into their final pentameric configuration is not known. The artificial intelligence program AlphaFold2 recently predicted structures for NACHO and RIC-3. NACHO is highly conserved in sequence and structure across species, but RIC-3 is not. This review ponders how different intrinsically disordered RIC-3 isoforms from C. elegans to humans interact with α7 nAChR subunits despite having little sequence homology across RIC-3 species. Two models from the literature about how RIC-3 assists α7 nAChR assembly are evaluated considering recent structural information about the receptor and its chaperones

GTS-21 has cell-specific anti-inflammatory effects independent of α7 nicotinic acetylcholine receptors.

α7 Nicotinic acetylcholine receptors (nAChRs) reportedly reduce inflammation by blocking effects of the important pro-inflammatory transcription factor, nuclear factor kappa-light chain-enhancer of B cells (NFκB). The α7 nAChR partial agonist GTS-21 reduces secretion of pro-inflammatory cytokines including interleukin-6 (IL6) and tumor-necrosis factor (TNF) in models of endotoxemia and sepsis, and its anti-inflammatory effects are widely ascribed to α7 nAChR activation. However, mechanistic details of α7 nAChR involvement in GTS-21 effects on inflammatory pathways remain unclear. Here, we investigate how GTS-21 acts in two cell systems including the non-immune rat pituitary cell line GH4C1 expressing an NFκB-driven reporter gene and cytokine secretion by ex vivo cultures of primary mouse macrophages activated by lipopolysaccharide (LPS). GTS-21 does not change TNF-stimulated NFκB signaling in GH4C1 cells expressing rat α7 nAChRs, suggesting that GTS-21 requires additional unidentified factors besides α7 nAChR expression to allow anti-inflammatory effects in these cells. In contrast, GTS-21 dose-dependently suppresses LPS-induced IL6 and TNF secretion in primary mouse macrophages endogenously expressing α7 nAChRs. GTS-21 also blocks TNF-induced phosphorylation of NFκB inhibitor alpha (IκBα), an important intermediary in NFκB signaling. However, α7 antagonists methyllycaconitine and α-bungarotoxin only partially reverse GTS-21 blockade of IL6 and TNF secretion. Further, GTS-21 significantly inhibited LPS-induced IL6 and TNF secretion in macrophages isolated from knockout mice lacking α7 nAChRs. These data indicate that even though a discrete component of the anti-inflammatory effects of GTS-21 requires expression of α7 nAChRs in macrophages, GTS-21 also has anti-inflammatory effects independent of these receptors depending on the cellular context

Anti-inflammatory effects of astroglial α7 nicotinic acetylcholine receptors are mediated by inhibition of the NF-κB pathway and activation of the Nrf2 pathway

Abstract Background α7 nicotinic acetylcholine receptors (nAChRs) are widely distributed throughout the central nervous system and are reported to have neuroprotective properties. α7 nAChRs are expressed on astrocytes, which are key regulators of neuroinflammation and oxidative stress in several neurodegenerative diseases. However, the anti-inflammatory and antioxidant properties of astroglial α7 nAChRs are not well studied. Therefore, we evaluated the role of astroglial α7 nAChR activation in neuroinflammation. Methods Anti-inflammatory and antioxidant effects of α7 nAChR activation were evaluated in an in vitro mouse model of neuroinflammation using lipopolysaccharide (LPS) in primary astrocyte cultures. α7 nAChR anti-inflammatory effects on the NF-κB pathway were evaluated using ELISA, gene expression analysis, immunofluorescence, and western blotting. Antioxidant effect of α7 nAChR activation on expression profiles of canonical Nrf2 target genes was examined by quantitative PCR and western blotting. The role of the Nrf2 pathway in α7 nAChR-mediated anti-inflammatory response was evaluated using Nrf2 knockout astrocytes. Brain ex vivo NF-κB luciferase signals were evaluated after treatment with an α7 nAChR agonist in lipopolysaccharide (LPS)-injected NF-κB luciferase reporter mouse model. Results Astrocytes treated with the α7 nAChR partial agonist (GTS21) showed significantly reduced LPS-mediated secretion of inflammatory cytokines and this effect was reversed by the α7 nAChR antagonist methyllycaconitine (MLA) and by knockdown of α7 nAChR expression with a short hairpin RNA. Further, α7 nAChR activation blocked LPS-mediated NF-κB nuclear translocation indicating that the observed anti-inflammatory effect may be mediated through inhibition of the NF-κB pathway. Treatment with GTS21 also upregulated canonical Nrf2 antioxidant genes and proteins suggesting antioxidant properties of α7 nAChR in astrocytes. Using an astrocyte conditioned media approach, we demonstrated reduction in neuronal apoptosis when astrocytes were pretreated with GTS21. Finally, in an in vivo neuroinflammation model using LPS in NF-κB luciferase reporter mice, we demonstrated reduction in LPS-induced NF-κB activity and pro-inflammatory cytokines with GTS21 treatment in brain tissue. Conclusion Our results suggest that activating astroglial α7 nAChRs may have a role in neuroprotection by decreasing inflammation and oxidative stress, and therefore could have therapeutic implication for disease modifying treatments of neurodegenerative diseases

Investigating the role of protein folding and assembly in cell-type dependent expression of alpha7 nicotinic receptors using a green fluorescent protein chimera

To test the hypothesis that cell-dependent expression of α7 receptors is due to differences in protein folding or assembly, we constructed a chimeric rat α7 subunit with green fluorescent protein (GFP) at the receptor C-terminal. Expression of α7-GFP in Xenopus oocytes resulted in currents that were indistinguishable from wild type receptors but were only 33% of control. 125I-α-bungarotoxin (αBGT) binding at the oocyte surface was reduced to 23% of wild type. Transfection of α7-GFP into GH4C1 cells produced fluorescence that was less intense than GFP alone, but showed significant α-BGT binding compared to transfection with GFP. In contrast, α7-GFP transfection in SH-EP1, HEK293 and CHO-CAR cells produced fluorescence without αBGT binding. Flow cytometry of cells transfected with α7-GFP indicated fluorescence in both SH-EP1 and GH4C1 cells, but surface toxin binding sites and sites immunoprecipitated using anti-GFP antibodies were undetectable in SH-EP1 cells, suggesting a problem in folding/assembly rather than trafficking. Surprisingly, integrated fluorescence intensities in GH4C1 cells transfected with α7-GFP did not correlate with amounts of cell surface or immunoprecipitable αBGT binding. Therefore, GFP folding at the C-terminal of the α7-GFP chimera is cell-line independent, but toxin binding is highly cell-line dependent, suggesting that if altered protein folding is involved in the cell-type dependence of α7 receptor expression, the phenomenon is restricted to specific protein domains. Further, C-terminal GFP-labeled α7 receptors decreased the efficiency of folding/assembly not only of chimeric subunits, but also wild-type subunits, suggesting that the C-terminal is an important domain for α7 receptor assembly.Supported by the Ministry of Education and Science of Spain and FEDER (SAF2005-00534 and SAF2005-02045), by NIH NS22472, and by an RSDF grant from Northeastern University.Peer Reviewe