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 Signaling,Neural Excitability,and Epilepsy

Epilepsy is a common brain disorder characterized by recurrent epileptic seizures with neuronal hyperexcitability. Apart from the classical imbalance between excitatory glutamatergic transmission and inhibitory γ-aminobutyric acidergic transmission, cumulative evidence suggest that cholinergic signaling is crucially involved in the modulation of neural excitability and epilepsy. In this review, we briefly describe the distribution of cholinergic neurons, muscarinic, and nicotinic receptors in the central nervous system and their relationship with neural excitability. Then, we summarize the findings from experimental and clinical research on the role of cholinergic signaling in epilepsy. Furthermore, we provide some perspectives on future investigation to reveal the precise role of the cholinergic system in epilepsy.     

Fluorescence Studies of the Acetylcholine Receptor: Structure and Dynamics in Membranes and Cells

The nicotinic acetylcholine receptor (AChR) is the archetype member of the superfamily of ligand-gated ion channels that mediate fast intercellular communication in response to endogenous neurotransmitters. Here I review a series of biophysical studies on the AChR protein, with particular focus on the interactions of the macromolecule with its lipid microenvironment. Fluorescence recovery after photobleaching and phosphorescence anisotropy studies of the membrane-embedded AChR have contributed to our understanding of the translational and rotational dynamics of this protein in synthetic lipid bilayers and in the native membrane. Electron spin resonance studies led to the discovery of a lipid fraction in direct contact with the AChR with rotational dynamics 50-fold slower than that of the bulk lipids. This lipid belt region around the AChR molecule has since been intensively studied with the aim to define its possible role in the modulation of receptor function. The polarity and molecular dynamics of solvent dipoles—mainly water—in the vicinity of the lipids in the AChR membrane have been studied exploiting the amphiphilic fluorescent probe Laurdan's exquisite sensitivity to the phase state of the membrane, and Förster-type resonance energy transfer (FRET) was introduced to characterize the receptor-associated lipid microenvironment. FRET was used to discriminate between the bulk lipid and the lipid belt region in the vicinity of the protein. Further refinement of this topographical information was provided by the parallax method using phospholipid spin labels. The AChR-vicinal lipid is in a liquid-ordered phase and exhibits a higher degree of order than the bulk bilayer lipid. Changes in FRET efficiency induced by fatty acids, phospholipid, and cholesterol also led to the identification of discrete sites for these lipids on the AChR protein. I also illustrate the extension of Laurdan fluorescence studies to intact living cells heterologously expressing AChR in a brief section devoted to recent studies using two-photon fluorescence microscopy. The spatial resolution afforded by the two-photon optical sectioning of the cell in combination with the advantageous spectroscopic properties of Laurdan are exploited to obtain information on the physical state of the lipid environment of the membrane. Finally, the application of site-specific labeling and steady-state fluorescence spectroscopy to probe the location of AChR membrane-embedded domains is illustrated. The topography of the pyrene-labeled Cys residues in transmembrane domains M1, M4, M1, and M4 with respect to the membrane was determined by differential fluorescence quenching with lipid-resident spin-labeled probes. Cys residues were found to lie in a shallow position. For M4 segments, this is compatible with a linear -helical structure, but not so for M1, for which classical models locate Cys residues at the center of the hydrophobic stretch. The transmembrane topography of M1 can be rationalized on the basis of the presence of a substantial amount of nonhelical structure and/or of kinks attributable to the occurrence of the evolutionarily conserved proline residues. The latter is a striking feature of M1 in the AChR and all members of the rapid ligand-gated ion channel superfamily.     

WHOLE-CELL CLAMP STUDY OF XENOPUS EMBRYONIC CHOLINERGIC NEURONS

Whole-cell clamped myoballs are placed into direct visible contact with the growth cones of isolated neurons in embryonic Xenopus culture to serve as probe of acetylchollne (AcCHo) release in order to determine whether these neurons are cholinergic or not. Using a GQ-seal, whole-cell recording method, the electrophysiological properties of these identified cholinergic neurons are studied. It is found that these embryonic neurons, like adult frog motor neurons, exhibit repetitive firings in a certain embryonic developing stage. A development of repetitive firings is observed simultaneously. Tracing the development of one neuron, we find that the development of repetitive firing is completed at the 48th h after fertilization. Tetrodotoxin (TTX) which blocks Na~+ channels can abolish all firings; and tetraethyl ammonium chloride (TEA), the blocker of K~+ channels, reverses this development, i. e. it makes the repetitive firings disappear again. These data show that the nature of the development of repetiti     

An Inside Job: Molecular Determinants for Postsynaptic Localization of Nicotinic Acetylcholine Receptors

Nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic transmission at neuromuscular and autonomic ganglionic synapses in the peripheral nervous system. The postsynaptic localization of muscle ((α1)₂β1γδ) and neuronal ((α3β4)₂β4) nicotinic receptors at these synapses is mediated by interactions between the nAChR intracellular domains and cytoplasmic scaffolding proteins. Recent high resolution structures and functional studies provide new insights into the molecular determinants that mediate these interactions. Surprisingly, they reveal that the muscle nAChR binds 1–3 rapsyn scaffolding molecules, which dimerize and thereby form an interconnected lattice between receptors. Moreover, rapsyn binds two distinct sites on the nAChR subunit cytoplasmic loops; the MA-helix on one or more subunits and a motif specific to the β subunit. Binding at the latter site is regulated by agrin-induced phosphorylation of βY390, and increases the stoichiometry of rapsyn/AChR complexes. Similarly, the neuronal nAChR may be localized at ganglionic synapses by phosphorylation-dependent interactions with 14-3-3 adaptor proteins which bind specific motifs in each of the α3 subunit cytoplasmic loops. Thus, postsynaptic localization of nAChRs is mediated by regulated interactions with multiple scaffolding molecules, and the stoichiometry of these complexes likely helps regulate the number, density, and stability of receptors at the synapse.     

The Influence of CB2-Receptor Ligands on the Memory-Related Responses in Connection with Cholinergic Pathways in Mice in the Passive Avoidance Test

Background: Dysfunction of the cholinergic system is associated with the development of Alzheimer’s disease (AD). One of the new possible strategies for the pharmacological modulation of memory-related problems typical of AD, is connected with the endocannabinoid system (ECS) and the cannabinoid (CB: CB1 and CB2) receptors. Methods: The aim of the study was to determine the influence of the selective CB2 receptor ligands: agonist (JWH 133) and antagonist (AM 630) on different stages of memory and learning in mice, in the context of their interaction with cholinergic pathways. To assess and understand the memory-related effects in mice we used the passive avoidance (PA) test. Results: We revealed that co-administration of non-effective dose of JWH 133 (0.25 mg) or AM 630 (0.25 mg/kg) with the non-effective dose of cholinergic receptor agonist - nicotine (0.05 mg/kg) enhanced cognition in the PA test in mice; however, an acute injection of JWH 133 (0.25 mg/kg) or AM 630 (0.25 mg/kg) had no influence on memory enhancement induced by the effective dose of nicotine (0.1 mg/kg). Co-administration of JWH 133 (0.25 mg) or AM 630 (0.25 mg/kg) with the effective dose of the cholinergic receptor antagonist scopolamine (1 mg/kg) attenuated the scopolamine-induced memory impairment in the PA test in mice. Conclusion: Our experiments have shown that CB2 receptors participate in the modulation of memory-related responses, especially those in which cholinergic pathways are implicated.     

Neuroprotective Effect of Yucca schidigera Roezl ex Ortgies Bark Phenolic Fractions,Yuccaol B and Gloriosaol A on Scopolamine-Induced Memory Deficits in Zebrafish

Y. schidigera contains a number of unusual polyphenols, derivatives of resveratrol and naringenin, called spiro-flavostilbenoids, which have potent in vitro anti-inflammatory, antioxidant, and moderate cholinesterase inhibitory activities. To date, these compounds have not been tested in vivo for the treatment of neurodegenerative diseases. The aim of the present study was to evaluate the effects of both single spiro-flavostilbenoids (yuccaol B and gloriosaol A) and phenolic fractions derived from Y. schidigera bark on scopolamine-induced anxiety and memory process deterioration using a Danio rerio model. Detailed phytochemical analysis of the studied fractions was carried out using different chromatographic techniques and Nuclear Magnetic Resonance (NMR). The novel tank diving test was used as a method to measure zebrafish anxiety, whereas spatial working memory function was assessed in Y-maze. In addition, acetylcholinesterase/butyrylcholinesterase (AChE/BChE) and 15-lipooxygenase (15-LOX) inhibition tests were performed in vitro. All pure compounds and fractions under study exerted anxiolytic and procognitive action. Moreover, strong anti-oxidant capacity was observed, whereas weak inhibition towards cholinesterases was found. Thus, we may conclude that the observed behavioral effects are complex and result rather from inhibition of oxidative stress processes and influence on cholinergic muscarinic receptors (both 15-LOX and scopolamine assays) than effects on cholinesterases. Y. schidigera is a source of substances with desirable properties in the prevention and treatment of neurodegenerative diseases.     

Cholinergic Chemotransmission and Anesthetic Drug Effects at the Carotid Bodies

General anesthesia is obtained by administration of potent hypnotics, analgesics and muscle relaxants. Apart from their intended effects (loss of consciousness, pain relief and muscle relaxation), these agents profoundly affect the control of breathing, in part by an effect within the peripheral chemoreflex loop that originates at the carotid bodies. This review assesses the role of cholinergic chemotransmission in the peripheral chemoreflex loop and the mechanisms through which muscle relaxants and hypnotics interfere with peripheral chemosensitivity. Additionally, consequences for clinical practice are discussed.     

Characteristics of evodiamine-exerted stimulatory effects on rat jejunal contractility

This study was designed to characterise the effects of evodiamine on intestinal contractility and reveal the correlated mechanisms. Evodiamine (2.5–80.0 μM) increased normal jejunal contractility and jejunal hypocontractility established under a variety of experimental conditions. Evodiamine-exerted stimulatory effects were blocked by the L-type Ca²⁺ channel blocker nifedipine or abolished in the Ca²⁺-free assay condition. The stimulatory effects of evodiamine on jejunal contractility were partially blocked in the presence of neurotoxin tetrodotoxin or endogenous acetylcholine synthesis blocker hemicholinium-3 or muscarinic receptor antagonist atropine, respectively. Evodiamine-exerted stimulatory effects were blocked by c-kit receptor tyrosine kinase inhibitor imatinib. Evodiamine increased myosin phosphorylation in jejunal smooth muscle of constipation-prominent rats. These results showed that evodiamine-exerted stimulatory effects on jejunal segments are Ca²⁺-dependent, need the presence of interstitial cell of Cajal, requirement of cholinergic neuron and correlate with increased myosin phosphorylation, implicating the potential value of evodiamine in relieving hypo-motility disorders.