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.

The Redox Biology of Excitotoxic Processes: The NMDA Receptor, TOPA Quinone, and the Oxidative Liberation of Intracellular Zinc.

This special issue of Frontiers in Neuroscience-Neurodegeneration celebrates the 50th anniversary of John Olney's seminal work introducing the concept of excitotoxicity as a mechanism for neuronal cell death. Since that time, fundamental research on the pathophysiological activation of glutamate receptors has played a central role in our understanding of excitotoxic cellular signaling pathways, leading to the discovery of many potential therapeutic targets in the treatment of acute or chronic/progressive neurodegenerative disorders. Importantly, excitotoxic signaling processes have been found repeatedly to be closely intertwined with oxidative cellular cascades. With this in mind, this review looks back at long-standing collaborative efforts by the authors linking cellular redox status and glutamate neurotoxicity, focusing first on the discovery of the redox modulatory site of the N-methyl-D-aspartate (NMDA) receptor, followed by the study of the oxidative conversion of 3,4-dihydroxyphenylalanine (DOPA) to the non-NMDA receptor agonist and neurotoxin 2,4,5-trihydroxyphenylalanine (TOPA) quinone. Finally, we summarize our work linking oxidative injury to the liberation of zinc from intracellular metal binding proteins, leading to the uncovering of a signaling mechanism connecting excitotoxicity with zinc-activated cell death-signaling cascades.

Why Does Knocking Out NACHO, But Not RIC3, Completely Block Expression of α7 Nicotinic Receptors in Mouse Brain?

Alpha7 nicotinic acetylcholine receptors (α7nAChRs) are interesting not only because of their physiological effects, but because this receptor requires chaperones to traffic to cell surfaces (measured by alpha-bungarotoxin [αBGT] binding). While knockout (KO) animals and antibodies that react across species exist for tmem35a encoding the protein chaperone NACHO, commercially available antibodies against the chaperone RIC3 that allow Western blots across species have not been generally available. Further, no effects of deleting RIC3 function (ric3 KO) on α7nAChR expression are reported. Finally, antibodies against α7nAChRs have shown various deficiencies. We find mouse macrophages bind αBGT but lack NACHO. We also report on a new α7nAChR antibody and testing commercially available anti-RIC3 antibodies that react across species allowing Western blot analysis of in vitro cultures. These antibodies also react to specific RIC3 splice variants and single-nucleotide polymorphisms. Preliminary autoradiographic analysis reveals that ric3 KOs show subtle αBGT binding changes across different mouse brain regions, while tmem35a KOs show a complete loss of αBGT binding. These findings are inconsistent with effects observed in vitro, as RIC3 promotes αBGT binding to α7nAChRs expressed in HEK cells, even in the absence of NACHO. Collectively, additional regulatory factors are likely involved in the in vivo expression of α7nAChRs.

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.

Metabolic studies of synaptamide in an immortalized dopaminergic cell line.

INTRODUCTION: Synaptamide, the N-acylethanolamine of docosahexaenoic acid (DHA), is structurally similar to the endocannabinoid N-arachidonoylethanolamine, anandamide. It is an endogenous ligand at the orphan G-protein coupled receptor 110 (GPR110; ADGRF1), and induces neuritogenesis and synaptogenesis in hippocampal and cortical neurons, as well as neuronal differentiation in neural stem cells. PURPOSE: Our goal was to characterize the metabolic fate (synthesis and metabolism) of synaptamide in a dopaminergic cell line using immortalized fetal mesencephalic cells (N27 cells). Both undifferentiated and differentiating N27 cells were used in this study in an effort to understand synaptamide synthesis and metabolism in developing and adult cells. METHODS: Radiotracer uptake and hydrolysis assays were conducted in N27 cells incubated with [1-14C]DHA or with one of two radioisotopomers of synaptamide: [α,ß-14C2]synaptamide and [1-14C-DHA]synaptamide. RESULTS: Neither differentiated nor undifferentiated N27 cells synthesized synaptamide from radioactive DHA, but both rapidly incorporated radioactivity from exogenous synaptamide into membrane phospholipids, regardless of which isotopomer was used. Pharmacological inhibition of fatty acid amide hydrolase (FAAH) reduced formation of labeled phospholipids in undifferentiated but not differentiated cells. CONCLUSIONS: In undifferentiated cells, synaptamide uptake and metabolism is driven by its enzymatic hydrolysis (fatty acid amide hydrolase; FAAH), but in differentiating cells, the process seems to be FAAH independent. We conclude that differentiated and undifferentiated N27 cells utilize synaptamide via different mechanisms. This observation could be extrapolated to how different mechanisms may be in place for synaptamide uptake and metabolism in developing and adult dopaminergic cells.

Evaluating Commercially Available Antibodies for Rat α7 Nicotinic Acetylcholine Receptors.

Alpha7 nicotinic acetylcholine receptors (α7 nAChRs) are important drug targets in neurological disorders and inflammation, making their detection and localization by validated antibodies highly desirable. However, tests in knockout animals raised questions about specificity of antibodies to mouse α7 nAChRs. To date, methods for validating antibodies for rat or human α7 nAChR have not been reported. We developed a gel-shift assay for western blots using GH4C1 cells expressing either native rat receptors or α7 nAChR-green fluorescent protein (GFP) chimeras to evaluate seven commercially available α7 nAChR antibodies. Blots with anti-GFP antibody detected GFP or α7 nAChR-GFP expressed in GH4C1 cells, and 125I-α-bungarotoxin binding and RNA analysis demonstrated α7 nAChR expression. Validated samples were used to evaluate α7 nAChR antibodies by western blot and immunofluorescence studies. These methods confirmed that two of seven α7 nAChR antibodies identify gel-shifts for α7 nAChR/nAChR-GFP but only one antibody demonstrated low background and significant immunofluorescence differences between wild-type and α7 nAChR expressing GH4C1 cells. However, that polyclonal antibody displayed lot-to-lot variability. Our findings suggest that careful validation methods are required for all α7 nAChR receptor species and antibody lots and that the gel-shift assay may allow for relatively rapid antibody screening.

Jasmonate-dependent alkaloid biosynthesis in Catharanthus Roseus hairy root cultures is correlated with the relative expression of Orca and Zct transcription factors.

The effects of methyl jasmonate (MJ) dosage on terpenoid indole alkaloid (TIA) biosynthesis in Catharanthus roseus are correlated with the relative levels of specific MJ-responsive transcription factors. In this study, the expression of transcription factors (Orca, Zct, Gbf, Myc2, At-hook, and Wrky1), TIA pathway genes (G10h, Tdc, Str, and Sgd), and TIA metabolites (secologanin, strictosidine, and tabersonine) were investigated in C. roseus hairy root cultures elicited with a range of MJ dosages (0-1,000 µM) during mid-exponential growth. The highest production of TIA metabolites occurs at 250 µM MJ, increasing by 150-370% compared with untreated controls. At this MJ dosage, the expression of the transcriptional activators (Orca) is dramatically increased (29-40 fold) while the levels of the transcriptional repressors (Zct) remain low (2-7 fold). Simultaneously, the expression of genes coding for key enzymes involved in TIA biosynthesis increases by 8-15 fold. In contrast, high MJ dosages (1,000 µM) inhibit the production of TIA metabolites. This dosage is correlated with elevated expression levels of Zct (up to 40-fold) relative to Orca (13-19-fold) and minimal induction of the TIA biosynthetic genes (0-6 fold). The significant changes in the expression of Orca and Zct with MJ dosage do not correspond to changes in the expression of the early-response transcription factors (AT-hook, Myc2, and Wrky1) believed to regulate Orca and Zct. In summary, these observations suggest that the dependence of alkaloid production on MJ dosage in C. roseus may be partly mediated through the relative levels of Orca and Zct family transcription factors.

Cell-specific effects on surface α7 nicotinic receptor expression revealed by over-expression and knockdown of rat RIC3 protein.

We tested whether surface α7 nicotinic acetylcholine receptor expression is dependent on an endogenous chaperone named Resistance to Inhibitors of Cholinesterase 3 (RIC3) by comparing RIC3 protein in rat GH4C1 and human SH-EP1 cells, which express strikingly different surface receptor levels following α7 transfection. Cloned rat RIC3 exists in at least two isoforms because of an ambiguous splice site between exons 4 and 5. Both rat isoforms permit surface α7 expression in SH-EP1 and human embryonic kidney (HEK) cells measured by α-bungarotoxin binding. Contrary to expectations, endogenous RIC3 protein expression determined by immunoblots did not differ between untransfected GH4C1 or SH-EP1 cells. siRNA against rat RIC3 exon 4 and shRNA against exons 2, 5 and 6 knocked down transfected rat RIC3 expression in SH-EP1 cells and simultaneously blocked toxin binding. However, no RNAi construct blocked binding when co-transfected with α7 into GH4C1 cells. shRNA against rat exons 2 and 5 knocked down rat RIC3 protein transfected into GH4C1 cells with a time course suggesting a protein half-life of a few days. These results suggest GH4C1 cells may possess unknown chaperone(s) allowing high surface α7 expression in the absence of known RIC3 splice variants.

α4ß2 nicotinic receptors partially mediate anti-inflammatory effects through Janus kinase 2-signal transducer and activator of transcription 3 but not calcium or cAMP signaling.

Despite evidence that smoking confers protection against neurological disorders, how and whether specific nicotinic receptor subtypes are involved is unknown. We reported previously that nicotine suppresses constitutive nuclear factor κB (NF-κB) activity and thereby proinflammatory cytokine (PIC) production in SHEP1 cells stably transfected with α4ß2 nicotinic receptors. Here, we report the anti-inflammatory effects of nicotine pretreatment in lipopolysaccharide (LPS)-stimulated SHEP1 cells. Nicotine (100-300 nM, concentrations found in smoker's blood) blocked LPS-induced NF-κB translocation and production of PICs interleukin (IL)-1ß and IL-6 but only partially blocked inhibitor of nuclear factor-κBα (IκBα) phosphorylation. These effects were exclusively in cells transfected with α4ß2 receptors but not in wild types. The cell-permeable calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester, the adenylate cyclase stimulant forskolin, and a specific protein kinase A (PKA) inhibitor PKI 14-22-amide failed to block the effect of nicotine on LPS-induced NF-κB translocation and IκBα phosphorylation. However, the effects of nicotine on NF-κB activity were significantly blocked by the highly specific janus kinase 2 (JAK2) inhibitor α-cyano-(3,4-dihydroxy)-N-benzylcinnamide (AG-490) and the signal transducer and activator of transcription 3 (STAT3) inhibitor 2-hydroxy-4-[[[[(4-methylphenyl)sulfonyl]oxy]acetyl]amino]-benzoic acid (NSC74859). These findings reveal a calcium- and cAMP-PKA-independent signaling cascade and suggest a role for JAK2-STAT3 transduction in α4ß2-mediated attenuation of LPS-induced inflammation. Anti-inflammatory effects of nicotine may therefore be mediated through α4ß2 receptors, the predominant high-affinity binding sites for nicotine in the central nervous system, in addition to the better-established α7 receptors.

Assessing the limitations to terpenoid indole alkaloid biosynthesis in Catharanthus roseus hairy root cultures through gene expression profiling and precursor feeding.

The production of pharmaceutically important terpenoid indole alkaloids (TIAs) from Catharanthus roseus is partly regulated at the transcriptional level. In this study, limitations in TIA biosynthesis from C. roseus hairy root cultures were assessed through gene expression profiling and precursor feeding. The transcript levels of key TIA pathway genes (G10h, Tdc, Str, and Sgd) and metabolite levels associated with the TIA pathway (tryptamine, loganin, secologanin, strictosidine, ajmalicine, serpentine, and tabersonine) were monitored using quantitative RT-PCR and HPLC, respectively. In cultures elicited with methyl jasmonate (250 microM MeJA on day 21), G10h, Tdc, Str, and Sgd expression increased by 9.1, 3.1, 6.7, and 8.3-fold, respectively, after 24 h. Up-regulation of gene expression was followed by a 160, 440, and 420% increase in strictosidine, ajmalicine, and tabersonine levels, respectively, after 5 days. Precursors loganin, tryptamine, or their combination were fed to noninduced and MeJA-induced cultures to complement the above studies. TIA production was not significantly enhanced in either noninduced or MeJA-induced cultures with precursor feeding. In noninduced cells, steps downstream of loganin and tryptamine were limiting (SLS, STR, or SGD) because either loganin or tryptamine accumulated in the cells with precursor feeding. These bottlenecks were partly overcome in MeJA-induced cultures as the expression of Str and Sgd genes and TIA production increased. However, secologanin accumulated in MeJA-induced cultures with precursor feeding, suggesting that STR was likely limiting under MeJA-induced conditions.

Gene regulation of alpha4beta2 nicotinic receptors: microarray analysis of nicotine-induced receptor up-regulation and anti-inflammatory effects.

alpha4beta2 Nicotinic acetylcholine receptors play an important role in the reward pathways for nicotine. We investigated whether receptor up-regulation of alpha4beta2 nicotinic acetylcholine receptors involves expression changes for non-receptor genes. In a microarray analysis, 10 muM nicotine altered expression of 41 genes at 0.25, 1, 8 and 24 h in halpha4beta2 SH-EP1 cells. The maximum number of gene changes occurred at 8 h, around the initial increase in (3)[H]-cytisine binding. Quantitative RT-PCR corroborated gene induction of endoplasmic reticulum proteins CRELD2, PDIA6, and HERPUD1, and suppression of the pro-inflammatory cytokines IL-1beta and IL-6. Nicotine suppresses IL-1beta and IL-6 expression at least in part by inhibiting NFkappaB activation. Antagonists dihydro-beta-erythroidine and mecamylamine blocked these nicotine-induced changes showing that receptor activation is required. Antagonists alone or in combination with nicotine suppressed CRELD2 message while increasing alpha4beta2 binding. Additionally, small interfering RNA knockdown of CRELD2 increased basal alpha4beta2 receptor expression, and antagonists decreased CRELD2 expression even in the absence of alpha4beta2 receptors. These data suggest that endoplasmic reticulum proteins such as CRELD2 can regulate alpha4beta2 expression, and may explain antagonist actions in nicotine-induced receptor up-regulation. Further, the unexpected finding that nicotine suppresses inflammatory cytokines suggests that nicotinic alpha4beta2 receptor activation promotes anti-inflammatory effects similar to alpha7 receptor activation.

Effects of P-Aminophenyl Dichloroarsine on Reduced High-affinity [3H]Nicotine Binding Sites from Chick Brain: A Covalent, Yet Reversible, Agent for Neuronal Nicotinic Receptors.

Neuronal nicotinic acetylcholine receptor (nAChR) alpha-subunits contain a conserved disulphide that is essential for function. Here, we have examined the effects of sulphydryl redox reagents on [3H]nicotine binding to chick brain nAChR immunoisolated with the monoclonal antibody mAb35. The disulphide reducing agent, dithiothreitol (DTT), inhibited [3H]nicotine binding [50% inhibitory concentration (IC50)=146 microM] but this effect was reversed (93 +/- 1.5%) by subsequent reoxidation with 1 mM dithio-bis(nitrobenzoic acid) (DTNB). The trivalent arsenical, p-aminophenyl dichloroarsine (APA), which reacts with pairs of spatially close sulphydryls, was a potent inhibitor of reoxidation by DTNB (IC50=35 nM). However, application of the 'anti-arsenical', 2,3-dimercaptopropane sulphonic acid (DMPS), restored agonist binding after APA treatment (50% effective concentration=120 microM). Paradoxically, DMPS was also found to be a potent oxidizing agent of these receptors. Affinity alkylation of reduced nAChRs with bromoacetylcholine (BAC; 100 microM) irreversibly blocked nicotine binding (>90%). We propose (but have not proven) that APA interacts with the cysteines homologous to Cys192 - 193 in Torpedo AChRs, since APA pretreatment of reduced neuronal receptors protected against irreversible BAC alkylation, as shown by subsequent reversal of DMPS (2 mM; 20 min). This study illustrates the potent and reversible nature of the arsenical's covalent interaction with an isolated nAChR and suggests that modified arsenicals could be useful nAChR probes.

Pharmacological and Biochemical Properties of Nicotinic Receptors from Chick Retina.

Previous work has established that functional nicotinic receptors in the chick retina are blocked by neuronal bungarotoxin (NBT), and that the binding of radio-iodinated NBT to retinal homogenates is displaced by nicotinic ligands. In the present study, we examined the desensitizing effects of agonists on nicotinically-mediated depolarizations recorded from chick retina. The concentrations of five agonists necessary to reduce the amplitude of these depolarizations by 50% were found to correlate closely with the concentrations of these same agonists previously found necessary to displace 50% of NBT binding. In addition, bromoacetylcholine (BAC), a selective affinity alkylating agent for the agonist binding site, irreversibly inactivated the functional responses of intact chick retina with an inhibiting concentration for 50% block (IC50) near 10-6 M, the same concentration of BAC that displaced 50% of labelled NBT binding from alkylated retinal homogenates. These data suggest that NBT acts at the receptor agonist binding site. Furthermore, this binding site has a relatively low affinity for agonists, in the micromolar range, even in the desensitized state. Multiple subtypes of nicotinic receptors are known to exist in neuronal tissue, and receptors that bind agonists in the nanomolar range have been detergent-solubilized and purified using monoclonal antibodies. Under similar conditions, detergent-solubilization of chick retinal homogenates interfere with the interaction between NBT and the low-affinity neuronal nicotinic receptors. These data suggest that the conditions used to purify high-affinity neuronal nicotinic receptors may denature the subtype(s) of neuronal receptors recognized by NBT.