Impact of a worker bee thoracic ganglion RIC-3 variant on the actions of acetylcholine and neonicotinoids on nicotinic receptors in Apis mellifera.

A transmembrane thioredoxin (TMX3) enables the functional expression of insect nicotinic acetylcholine receptors (nAChRs) in Xenopus laevis oocytes, while co-factors RIC-3 and UNC-50 regulate the receptor expression level. RIC-3 (resistant to inhibitors of cholinesterase 3) has been shown to diversify by its differential mRNA splicing patterns. How such diversity influences neonicotinoid sensitivity of nAChRs of beneficial insect species remains poorly understood. We have identified a RIC-3 variant expressed most abundantly in the thoracic ganglia of honeybee (Apis mellifera) workers and investigated its effects on the functional expression and pharmacology of Amα1/Amα8/Amß1 and Amα1/Amα2/Amα8/Amß1 nAChRs expressed in X. laevis oocytes. The AmRIC-3 enhanced the response amplitude to the acetylcholine (ACh) of these A. mellifera nAChRs when its cRNA was injected into oocytes at low concentrations but suppressed the ACh response amplitude at high concentrations. Co-expression of the AmRIC-3 had a minimal impact on the affinity of ACh, but changed the efficacy of imidacloprid and clothianidin, suggesting that the presence and the level of RIC-3 expression can affect the nAChR responses to ACh and neonicotinoids, depending on nAChR subunit composition in honeybees. © 2024 Society of Chemical Industry.

N-Glycosylation Deficiency in Transgene α7 nAChR and RIC3 Expressing CHO Cells Without NACHO.

The human neuronal nicotinic acetylcholine receptor α7 (nAChR) is an important target implicated in diseases like Alzheimer's or Parkinson's, as well as a validated target for drug discovery. For α7 nAChR model systems, correct folding and ion influx functions are essential. Two chaperones, resistance to inhibitors of cholinesterase 3 (RIC3) and novel nAChR regulator (NACHO), enhance the assembly and function of α7 nAChR. This study investigates the consequence of NACHO absence on α7 nAChR expression and function. Therefore, the sequences of human α7 nAChR and human RIC3 were transduced in Chinese hamster ovary (CHO) cells. Protein expression and function of α7 nAChR were confirmed by Western blot and voltage clamp, respectively. Cellular viability was assessed by cell proliferation and lactate dehydrogenase assays. Intracellular and extracellular expression were determined by in/on-cell Western, compared with another nAChR subtype by novel cluster fluorescence-linked immunosorbent assay, and N-glycosylation efficiency was assessed by glycosylation digest. The transgene CHO cell line showed expected protein expression and function for α7 nAChR and cell viability was barely influenced by overexpression. While intracellular levels of α7 nAChR were as anticipated, plasma membrane insertion was low. The glycosylation digest revealed no appreciable N-glycosylation product. This study demonstrates a stable and functional cell line expressing α7 nAChR, whose protein expression, function, and viability are not affected by the absence of NACHO. The reduced plasma membrane insertion of α7 nAChR, combined with incorrect matured N-glycosylation at the Golgi apparatus, suggests a loss of recognition signal for lectin sorting.

Unraveling the molecular interactions between α7 nicotinic receptor and a RIC3 variant associated with backward speech.

Recent work putatively linked a rare genetic variant of the chaperone Resistant to Inhibitors of acetylcholinesterase (RIC3) (NM_024557.4:c.262G > A, NP_078833.3:p.G88R) to a unique ability to speak backwards, a language skill that is associated with exceptional working memory capacity. RIC3 is important for the folding, maturation, and functional expression of α7 nicotinic acetylcholine receptors (nAChR). We compared and contrasted the effects of RIC3G88R on assembly, cell surface expression, and function of human α7 receptors using fluorescent protein tagged α7 nAChR and Förster resonance energy transfer (FRET) microscopy imaging in combination with functional assays and 125I-α-bungarotoxin binding. As expected, the wild-type RIC3 protein was found to increase both cell surface and functional expression of α7 receptors. In contrast, the variant form of RIC3 decreased both. FRET analysis showed that RICG88R increased the interactions between RIC3 and α7 protein in the endoplasmic reticulum. These results provide interesting and novel data to show that a RIC3 variant alters the interaction of RIC3 and α7, which translates to decreased cell surface and functional expression of α7 nAChR.

Two residues determine nicotinic acetylcholine receptor requirement for RIC-3.

Nicotinic acetylcholine receptors (N-AChRs) mediate fast synaptic signaling and are members of the pentameric ligand-gated ion channel (pLGIC) family. They rely on a network of accessory proteins in vivo for correct formation and transport to the cell surface. Resistance to cholinesterase 3 (RIC-3) is an endoplasmic reticulum protein that physically interacts with nascent pLGIC subunits and promotes their oligomerization. It is not known why some N-AChRs require RIC-3 in heterologous expression systems, whereas others do not. Previously we reported that the ACR-16 N-AChR from the parasitic nematode Dracunculus medinensis does not require RIC-3 in Xenopus laevis oocytes. This is unusual because all other nematode ACR-16, like the closely related Ascaris suum ACR-16, require RIC-3. Their high sequence similarity limits the number of amino acids that may be responsible, and the goal of this study was to identify them. A series of chimeras and point mutations between A. suum and D. medinensis ACR-16, followed by functional characterization with electrophysiology, identified two residues that account for a majority of the receptor requirement for RIC-3. ACR-16 with R/K159 in the cys-loop and I504 in the C-terminal tail did not require RIC-3 for functional expression. Mutating either of these to R/K159E or I504T, residues found in other nematode ACR-16, conferred a RIC-3 requirement. Our results agree with previous studies showing that these regions interact and are involved in receptor synthesis. Although it is currently unclear what precise mechanism they regulate, these residues may be critical during specific subunit folding and/or assembly cascades that RIC-3 may promote.

Deletion induced splicing in RIC3 drives nicotinic acetylcholine receptor regulation with implications for endoplasmic reticulum stress in human astrocytes.

Nicotinic acetylcholine receptor (nAChR) dysregulation in astrocytes is reported in neurodegenerative disorders. Modulation of nAChRs through agonists confers protection to astrocytes from stress but regulation of chaperones involved in proteostasis with pathological implications is unclear. Resistance to inhibitors of cholinesterase 3 (RIC3), a potential chaperone of nAChRs is poorly studied in humans. We characterized RIC3 in astrocytes derived from an isogenic wild-type and Cas9 edited "del" human iPSC line harboring a 25 bp homozygous deletion in exon2. Altered RIC3 transcript ratio due to deletion induced splicing and an unexpected gain of α7nAChR expression were observed in "del" astrocytes. Transcriptome analysis showed higher expression of neurotransmitter/G-protein coupled receptors mediated by cAMP and calcium/calmodulin-dependent kinase signaling with increased cytokines/glutamate secretion. Functional implications examined using tunicamycin induced ER stress in wild-type astrocyte stress model showed cell cycle arrest, RIC3 upregulation, reduction in α7nAChR membrane levels but increased α4nAChR membrane expression. Conversely, tunicamycin-treated "del" astrocytes showed a comparatively higher α4nAChR membrane expression and upsurged cAMP signaling. Furthermore, reduced expression of stress markers CHOP, phospho-PERK and lowered XBP1 splicing in western blot and qPCR, validated by proteome-based pathway analysis indicated lowered disease severity. Findings indicate (i) a complex RNA regulatory mechanism via exonic deletion induced splicing; (ii) RIC-3 as a disordered protein having contrasting effects on co-expressed nAChR subtypes under basal/stress conditions; and (iii) RIC3 as a potential drug target against ER stress in astrocytes for neurodegenerative/nicotine-related brain disorders. Cellular rescue mechanism through deletion induced exon skipping may encourage ASO-based therapies for tauopathies.

RIC3 variants are not associated with Parkinson's disease in large European, Latin American, or East Asian cohorts.

Parkinson's disease (PD) is a complex neurodegenerative disorder in which both rare and common genetic variants contribute to disease risk. Multiple genes have been reported to be linked to monogenic PD but these only explain a fraction of the observed familial aggregation. Rare variants in RIC3 have been suggested to be associated with PD in the Indian population. However, replication studies yielded inconsistent results. We further investigate the role of RIC3 variants in PD in European cohorts using individual-level genotyping data from 14,671 PD patients and 17,667 controls, as well as whole-genome sequencing data from 1,615 patients and 961 controls. We also investigated RIC3 using summary statistics from a Latin American cohort of 1,481 individuals, and from a cohort of 31,575 individuals of East Asian ancestry. We did not identify any association between RIC3 and PD in any of the cohorts. However, more studies of rare variants in non-European ancestry populations, in particular South Asian populations, are necessary to further evaluate the world-wide role of RIC3 in PD etiology.

Neuroinflammation Modulation via α7 Nicotinic Acetylcholine Receptor and Its Chaperone, RIC-3.

Nicotinic acetylcholine receptors (nAChRs) are widely expressed in or on various cell types and have diverse functions. In immune cells nAChRs regulate proliferation, differentiation and cytokine release. Specifically, activation of the α7 nAChR reduces inflammation as part of the cholinergic anti-inflammatory pathway. Here we review numerous effects of α7 nAChR activation on immune cell function and differentiation. Further, we also describe evidence implicating this receptor and its chaperone RIC-3 in diseases of the central nervous system and in neuroinflammation, focusing on multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Deregulated neuroinflammation due to dysfunction of α7 nAChR provides one explanation for involvement of this receptor and of RIC-3 in neurodegenerative diseases. In this review, we also provide evidence implicating α7 nAChRs and RIC-3 in neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD) involving neuroinflammation. Besides, we will describe the therapeutic implications of activating the cholinergic anti-inflammatory pathway for diseases involving neuroinflammation.

Cholinergic transmission in C. elegans: Functions, diversity, and maturation of ACh-activated ion channels.

Acetylcholine is an abundant neurotransmitter in all animals. Effects of acetylcholine are excitatory, inhibitory, or modulatory depending on the receptor and cell type. Research using the nematode C. elegans has made ground-breaking contributions to the mechanistic understanding of cholinergic transmission. Powerful genetic screens for behavioral mutants or for responses to pharmacological reagents identified the core cellular machinery for synaptic transmission. Pharmacological reagents that perturb acetylcholine-mediated processes led to the discovery and also uncovered the composition and regulators of acetylcholine-activated channels and receptors. From a combination of electrophysiological and molecular cellular studies, we have gained a profound understanding of cholinergic signaling at the levels of synapses, neural circuits, and animal behaviors. This review will begin with a historical overview, then cover in-depth current knowledge on acetylcholine-activated ionotropic receptors, mechanisms regulating their functional expression and their functions in regulating locomotion.

RIC3, the cholinergic anti-inflammatory pathway, and neuroinflammation.

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels having many functions including inflammation control, as part of the cholinergic anti-inflammatory pathway. Genome wide association studies implicated RIC3, a chaperone of nAChRs, in multiple sclerosis (MS), a neuroinflammatory disease. To understand the involvement of RIC3 in inflammatory diseases we examined its expression, regulation, and function in activated immune cells. Our results show that immune activation leads to dynamic changes in RIC3 expression, in a mouse model of MS and in human lymphocytes and macrophages. We also show similarities in the expression dynamics of RIC3 and CHRNA7, encoding for the α7 nAChR subunit. Homomeric α7 nAChRs were shown to mediate the anti-inflammatory effects of cholinergic agonists. Thus, similarity in expression dynamics between RIC3 and CHRNA7 is suggestive of functional concordance. Indeed, siRNA mediated silencing of RIC3 in a mouse macrophage cell line eliminates the anti-inflammatory effects of cholinergic agonists. Furthermore, we show increased average expression of RIC3 and CHRNA7 in lymphocytes from MS patients, and a strong correlation between expression levels of these two genes in MS patients but not in healthy donors. Together, our results are consistent with a role for RIC3 and for the mechanisms regulating its expression in inflammatory processes and in neuroinflammatory diseases.

Reanalysis of exome sequencing data of intellectual disability samples: Yields and benefits.

Recently, with the advancement in next generation sequencing (NGS) along with the improvement of bioinformatics tools, whole exome sequencing (WES) has become the most efficient diagnostic test for patients with intellectual disability (ID). This study aims to estimate the yield of a reanalysis of ID negative exome cases after data reannotation. Total of 50 data files of exome sequencing, representing 50 samples were collected. The inclusion criteria include ID phenotype, and previous analysis indicated a negative result (no abnormality detected). These files were pre-processed and reannotated using ANNOVAR tool. Prioritized variants in the 50 cases studied were classified into three groups, (1) disease-causative variants (2) possible disease-causing variants and (3) variants in novel genes. Reanalysis resulted in the identification of pathogenic/likely pathogenic variants in six cases (12%). Thirteen cases (26%) were classified as having possible disease-causing variants. Candidate genes requiring future functional studies were detected in seven cases (14%). Improvement in bioinformatics tools, update in the genetic databases and literature, and patients' clinical phenotype update were the main reasons for identification of these variants in this study.

Dual effects of insect nAChR chaperone RIC-3 on hybrid receptor: Promoting assembly on endoplasmic reticulum but suppressing transport to plasma membrane on Xenopus oocytes.

Resistance to inhibitors of cholinesterase (RIC) -3 promotes the maturation (folding and assembly) of neuronal nicotinic acetylcholine receptors (nAChRs) as a molecular chaperone. The modulation effects of RIC-3 on homomeric α7 nAChRs are always positive, but its effects on heteromeric subtypes are inconsistent among reports. In this study, five RIC-3 isoforms were identified from Locusta migratoria. Four isoforms showed obvious effects on hybrid receptor Locα1/rß2 expressed in Xenopus oocytes. As a representative, the co-expression of RIC-3v4 exhibited the decreased agonist responses (Imax) on oocytes, lower specific [3H]epibatidine binding (Bmax) on plasma membrane protein (PMP), and reduced subunit levels in PMP, which showed that the mature Locα1/rß2 on the plasma membrane was decreased by the co-expression of RIC-3. In contrast, the [3H]epibatidine binding and mature Locα1/rß2 levels in the endoplasmic reticulum membrane protein (ERMP) were much increased when co-expressing with RIC-3v4. The [3H]epibatidine binding and mature Locα1/rß2 levels in total membrane protein (TMP) gave the similar results as that in ERMP. Taking data together, the results showed that the co-expression of RIC-3 increased the mature Locα1/rß2 receptor levels on ER of Xenopus oocytes, but these mature receptors were mostly kept on ER and suppressed to transport to plasma membrane.

Genetic analysis of the RIC3 gene in Han Chinese patients with Parkinson's disease.

Parkinson's disease (PD) is the second-most common etiologically complex neurodegenerative disease. Genetic abnormalities are thought to play an important role in the development of PD. Recently, mutations in the resistance to inhibitors of cholinesterase 3 gene (RIC3) have been reported to cause autosomal-dominant PD in Indian population. To determine whether RIC3 gene coding variant(s) are associated with PD in Han Chinese population, the RIC3 gene coding region in 218 mainland Han Chinese patients with PD and the identified variants in 242 normal controls were examined using direct sequencing analysis. Four known single nucleotide variants (c.354C>A, p.L118L, rs10839976; c.389G>A, p.C130Y, rs55990541; c.403C>T, p.P135S, rs73411617; and c.1054G>A, p.D352N, rs11826236) were identified in the RIC3 gene coding region. No significant differences were observed in either genotypic or allelic distributions between the PD patients and the normal controls (all P>0.05) for these four variants. Haplotype analysis showed that the presence of haplotype A-G-C-G (rs10839976-rs55990541-rs73411617-rs11826236) was associated with a 0.764-fold decreased risk (P=0.049, OR=0.764, 95% CI=0.585-0.999) for PD, whereas the presence of haplotype C-A-C-A was associated with a 2.143-fold increased risk (P=0.039, OR=2.143, 95% CI=1.023-4.488) for PD. The findings indicate that four variants: rs10839976, rs55990541, rs73411617 and rs11826236 in the RIC3 gene coding region may play little or no role in the development of PD. Two RIC3 gene haplotypes of four variants: A-G-C-G, and C-A-C-A might relate to either protection against or increased susceptibility to PD in the Han Chinese population, respectively.

NACHO Mediates Nicotinic Acetylcholine Receptor Function throughout the Brain.

Neuronal nicotinic acetylcholine receptors (nAChRs) participate in diverse aspects of brain function and mediate behavioral and addictive properties of nicotine. Neuronal nAChRs derive from combinations of α and ß subunits, whose assembly is tightly regulated. NACHO was recently identified as a chaperone for α7-type nAChRs. Here, we find NACHO mediates assembly of all major classes of presynaptic and postsynaptic nAChR tested. NACHO acts at early intracellular stages of nAChR subunit assembly and then synergizes with RIC-3 for receptor surface expression. NACHO knockout mice show profound deficits in binding sites for α-bungarotoxin, epibatidine, and conotoxin MII, illustrating essential roles for NACHO in proper assembly of α7-, α4ß2-, and α6-containing nAChRs, respectively. By contrast, GABAA receptors are unaffected consistent with NACHO specifically modulating nAChRs. NACHO knockout mice show abnormalities in locomotor and cognitive behaviors compatible with nAChR deficiency and underscore the importance of this chaperone for physiology and disease associated with nAChRs.

RIC3 variants are not associated with Parkinson's disease in French-Canadians and French.

Variants in the RIC3 gene have recently been suggested as a novel cause of Parkinson's disease (PD). Herein, the entire RIC3 gene was sequenced in a French-Canadian and French sample series of 535 PD patients and 527 unaffected controls. The effect of single variants and the combined effect of variants were calculated. Sequence Kernel association tests (SKAT, SKAT-O) were done on the entire gene level, and on the different domains and exons of RIC3. A total of 28 common and rare variants were identified in patients and controls. No significant association was found between any variant and haplotype in RIC3 and PD, and there was no over-representation of RIC3 variants at the entire gene, domain, or exon levels in patients versus controls. Our results do not support a role for RIC3 mutations as a common cause of PD in the French-Canadian and French populations.

Role of the α7 Nicotinic Acetylcholine Receptor and RIC-3 in the Cholinergic Anti-inflammatory Pathway.

BACKGROUND: The nicotinic acetylcholine receptor (nAChR) gene family encodes for subunits of acetylcholine gated ion channels. These receptors are expressed widely and have many functions: They mediate excitation at neuro-muscular junctions. Nicotinic Acetylcholine Receptor: In the central nervous system nAChRs have been implicated in memory, cognition, and addiction. And in non-excitatory cells they regulate differentiation, proliferation and inflammatory responses. The CHRNA7 gene encodes for the α7 nAChR subunit that assembles into a homomeric receptor having unusual properties. It is expressed widely and has many functions atypical for nAChRs; specifically, in immune cells α7 is required for the anti-inflammatory effects of acetylcholine and has been implicated in inflammatory autoimmune diseases including Multiple Sclerosis (MS). Interestingly, although, α7 receptors are found at the outer membranes of immune cells, acetylcholine-dependent currents have not been recorded from these cells. Therefore, its mechanism of action in immune cells needs further evaluation. Maturation of α7 into functional ligand-gated channels in the plasma membrane is a complex process shown to depend on the ER-resident chaperone, RIC-3. Therefore, RIC-3 regulates functional expression of α7. RIC-3 like α7 is expressed in immune cells and has been implicated in MS. Thus, RIC-3 may regulate functional expression of α7 in immune cells. CONCLUSION: In this review we describe effects and mechanism of action of α7 nAChR and RIC-3 in the immune cholinergic system. Elucidating these mechanisms and the regulation of α7 and RIC-3 in the immune cholinergic system can pave the way for novel immunomodulatory agents, or towards extending the application of cholinergic agents.

RIC-3 expression and splicing regulate nAChR functional expression.

BACKGROUND: The nicotinic acetylcholine receptors form a large and diverse family of acetylcholine gated ion channels having diverse roles in the central nervous system. Maturation of nicotinic acetylcholine receptors is a complex and inefficient process requiring assistance from multiple cellular factors including RIC-3, a functionally conserved endoplasmic reticulum-resident protein and nicotinic acetylcholine receptor-specific chaperone. In mammals and in Drosophila melanogaster RIC-3 is alternatively spliced to produce multiple isoforms. RESULTS: We used electrophysiological analysis in Xenopus laevis oocytes, in situ hybridization, and quantitative real-time polymerase chain reaction assays to investigate regulation of RIC-3's expression and splicing and its effects on the expression of three major neuronal nicotinic acetylcholine receptors. We found that RIC-3 expression level and splicing affect nicotinic acetylcholine receptor functional expression and that two conserved RIC-3 isoforms express in the brain differentially. Moreover, in immune cells RIC-3 expression and splicing are regulated by inflammatory signals. CONCLUSIONS: Regulation of expression level and splicing of RIC-3 in brain and in immune cells following inflammation enables regulation of nicotinic acetylcholine receptor functional expression. Specifically, in immune cells such regulation via effects on α7 nicotinic acetylcholine receptor, known to function in the cholinergic anti-inflammatory pathway, may have a role in neuroinflammatory diseases.