CNR - Institute of Neuroscience CNR
Institute of Neuroscience
 

Project

The cholinergic anti-inflammatory pathway. The case of the nicotinic alpha7 acetylcholine receptor and its duplicate isoform

Inflammation is a physiological process essential for survival, but at the same time is also a major cause of human morbidity and mortality. This is a dramatic example in sepsis, characterized by the uncontrolled production of proinflammatory cytokines such as TNF, which is important in the inflammatory response early in infection control and promoter of the bacterial repair processes. In addition, the majority of neurodegenerative diseases such as Alzheimer's and Parkinson's disease, are associated with states of chronic inflammation, caused by an excessive activation of brain mononuclear phagocytic cells, called microglia, which normally have a role to ensure and support appropriate neuronal function. Recent studies have shown that immune system can be no longer regarded as entirely autonomous in its regulation. Neuronal activities, by means of neurotransmitters’ release, regulate the host’s inflammatory response against pathogens infection and injury. Recent experimental evidence has suggested a possible involvement of the parasympathetic nervous system in the control of inflammation via the vagus nerve, a mechanism called "the cholinergic anti-inflammatory pathway", according to which the afferent component of the vagus nerve carries information about the inflammatory processes that occur at the peripheral level. The efferent fibres of the same nerve, releasing acetylcholine (ACh), reduce the level of pro-inflammatory cytokines (produced by innate immune cells) via a reflex mechanism. This hypothesis is supported by a rat models of endotoxemia, where the electrical stimulation of the vagus nerve lead to a reduction in the levels of TNF alpha.

 

Various studies have shown that the α7 nicotinic receptor is a key element for the functioning of this pathway.

The nicotinic receptors are part of the ligand-gated ion channel receptor superfamily, located in the central nervous system as well as in the peripheral and the muscle fibers. These receptors are composed by five integral membrane subunits; to date nine α, four β, one γ , one δ and one ε subunits are known. These are assembled to form a large number of homo-and eteropentameric receptors with distinct structural and pharmacological properties.

The α7 nicotinic receptor is a homomeric receptor composed of five α7 subunits, with an high permeability to calcium ions. The gene encoding the α7 subunit (CHRNA7) is located on the long arm of chromosome 15 (15q13-q14 region) and is 138 Kb long. It is composed by ten exons that give raise to a transcript encoding a protein of about 57 kDa. Exons 1 to 6 encode the N-terminus extracellular domain, that contains the ACh ligand binding domain, exons 7 and 8 coding the first three transmembrane domains, while exons 9 and 10 code the cytoplasmatic loop, the fourh TM domain and the extracellular C-terminus. This receptor is widely distributed both in the central and peripheral nervous system. In the central nervous system is located at the pre-synaptic level, where it modulates neurotransmitters release and at the post-synaptic level where it is responsible for the fast excitatory post-synaptic potential, that leads to the membrane depolarization and the propagation of the stimulus. α7 signalling is also associated with cell survival and neuronal plasticity.

The α7 receptor is also expressed in non-neuronal cells, such as endothelial cells, lymphocytes, keratinocytes, astrocytes, microglial cells and macrophages. Experimental evidence suggests that the α7 receptor in these not excitable cells may play a role in processes such as proliferation, differentiation and cell migration through an autocrine and paracrine transmission mechanism. In human macrophages the activation of this receptor by nicotine is able to interfere with the release of TNF α, IL-1, IL-6 and HMGB1 induced by pro- inflammatory stimuli such as LPS. The importance of α7 (CHRNA7) in the cholinergic anti-inflammatory pathway seems to be confirmed by experiments in which blocking of translation with antisense oligonucleotides results in a normal release of TNF in macrophages treated with nicotine. Furthermore, knockout mice for the α7 gene have higher concentrations of TNF compared to wild-type following the administration of endotoxin, in addition, the macrophages of these animals are refractory to cholinergic agonists and electrical stimulation of the vagus does not cause a reduction levels of TNFα.

The CHRFAM7A gene

Recent studies in humans have shown that a portion of the CHRNA7 gene, from exon 5 to 10, is duplicated, with an identity greater than 99%. This partial duplication has fused with the duplicated exons A, B, C from the serine/threonine kinase ULK4 gene, mapping to 3p22.1, and exon D of unknown provenance (FAM7A gene). The acquisition of this hybrid gene appears to be a very recent event from the evolutionary point of view, because it appears only in humans. CHRFAM7A gene is located on chromosome 15 (15q13-q14 region), 1.6 Mb apart from CHRNA7 gene, in the direction of the centromere, and in the opposite orientation with respect to CHRNA7. The CHRFAM7A transcript has been detected in the hippocampus, in the cortex, in the corpus callosum, in the thalamus, in the cerebellum and in cells of the immune system, like lymphocytes and monocytes. The gene is present in one or two copies in more than 95% of the population. The absence of the CHRFAM7A gene, found in a small percentage of subjects, would represent a later deletion of the primary duplication event due to further recombination, while an alternative explanation consider the absence of CHRFAM7A gene as an ancestral sequence that did not undergo duplication of CHRNA7. Moreover, a polymorphic inverted variant has been described, that is in linkage disequilibrium with a 2 bp deletion in exon 6. Clinical studies have shown a significant association between this polymorphism and schizophrenia, although it is not clear how the deletion might cause functional changes in the protein. The CHRFAM7A transcript contains sequences encoding the exon 5-10 of the CHRNA7. Therefore, the resulting protein product would contain the transmembrane domain and the cytoplasmic loop of CHRNA7 subunit. Furthermore, the resulting receptor would lack the signal peptide and the ligand-binding domain for acetylcholine (encoded in CHRNA7 by exon 1-4). Exons D-A do not contain known ligand-binding domain or localization signals and the function of this protein is still unknown. The gene encodes 2 isoforms, generated by alternative "splicing" event of the same transcript, whose molecular weight is 46.2 kDa and 35.5 kDa, respectively. Experiments carried out on human primary leukocytes in order to characterize the function of this protein, showed peculiar pharmacological properties. Unlike the classical α7 nicotinic receptor, the CHRFAM7A gene product was unable to bind α-bungarotoxin and, moreover, was not able to evoke detectable currents in response to treatment with ACh or nicotine. Recently, our laboratory has shown that LPS treatment of a human leukaemic monocytic cell line (THP-1) down-regulated the expression of the CHRFAM7A gene, mainly by a transcriptional mechanism, reliant on the transcription factor NF-κB. This mechanism was confirmed in primary cultures of macrophages (Benfante et al., 2011) in a physiological process. Unlike the THP-1 cell line, where CHRFAM7A is the only isoform expressed, primary macrophages express both the conventional form of α7 (CHRNA7) and CHRFAM7A. Treatment with LPS induces the expression of the CHRNA7 gene, suggesting that in these cells heteromeric α7 receptors, consisting of both α7 subunit, could be formed. This receptor would have a lower capacity to respond to acetylcholine, because the subunit has no conventional binding site for the ligand. This suggests that the negative regulation of CHRFAM7A gene may be somehow involved in regulating levels of homomeric α7 nicotinic receptor on the membrane of macrophages and therefore the ability of these immune cells to respond to acetylcholine released from vagus nerve during an infection.

The same hypothesis, according to which the duplicated α7 subunit would act as a dominant negative regulator of the α7 nicotinic receptor, has been formulated independently in another laboratory, with experimental data obtained by means of co-injection into Xenopus oocytes of mRNA encoding the α7 subunit along with the CHRFAM7A isoform 1 mRNA. Electrophysiological recordings have shown that the current induced after stimulation with nicotine decreases in proportion to the decline in the ratio of mRNA injected (α7: α7dup mRNA).

The possibility of a regulatory role for CHRFAM7A in the localization and function of the conventional CHRNA7 receptor, and thus the ability to be able to interfere with a correct response to pro-inflammatory stimuli, makes this protein interesting as a regulator of the “cholinergic antiinflammatory pathway” in humans.

In order to get insight into the function of CHRFAM7A gene, the project aims to define:

  • the molecular mechanisms that direct the transcription of the duplicated form of the α7 nicotinic receptor and identify the elements responsive to pro-inflammatory stimuli. The identification of the regulatory region of the gene is under investigation. Once cloned upstream the luciferase reporter gene, the region will be functionally tested by means of transient transfection studies in neuronal and monocytic cell lines in order to identify promoter elements important for proper CHRFAM7A gene expression in different tissues. Gel-shift assay (EMSA) and chromatin immunoprecipitation (ChIP) experiments will allow us to identify the transcription factors involved. In particular, treatment of cells with LPS, will lead to the identification of responsive sites that mediate the repressive effect of NF-kB. In addition, by means of "RNA interference (siRNA) approach, directed against different subunits of NF-kB, we will identify the composition of NF-kB, which mediates the repression of the expression of CHRFAM7A after stimulation with pro- inflammatory stimuli.
  • The role of the CHRFAM7A isoforms in the regulation of CHRNA7 cellular localization.
  • whether chronic inflammatory diseases are associated with a dis-regulation of this mechanism, either due to the lack of the gene, but also to defects in the regulation of transcription of the gene itself. This would make CHRFAM7A a new pharmacological target for the development of therapies involving α7, especially since the use of α7 agonists is limited by the occurrence of side effects.

Publications

  • Benfante R, Antonini RA, De Pizzol M, Gotti C, Clementi F, Locati M, Fornasari D (2011) Expression of the ?7 nAChR subunit duplicate form (CHRFAM7A) is down-regulated in the monocytic cell line THP-1 on treatment with LPS. J. Neuroimmunol. 230:74-84.

Collaborations

  • Cecilia Gotti, CNR - Institute of Neuroscience, Milan
  • Massimo Locati, UNIMI, Istituto Humanitas, Milano

 

PI photo

Roberta Benfante

Contact information

email  E-mail

email  +39 02 503 16945

Participating staff

Diego Fornasari

Simona Di Lascio

Elena Saba

Valentina Alari