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Cholinergic control of inflammation
Molecular biology techniques. Transient transfection studies in neuronal and monocytic cell lines, luciferase reporter gene assay, Gel-shift assay (EMSA) and chromatin immunoprecipitation (ChIP), RNA interference (siRNA), western blot, immunocytochemistry, confocal microscopy.
The role of the of the nicotinic alpha7 acetylcholine receptor and its duplicate isoform in the cholinergic anti-inflammatory pathway
Inflammation is a physiological process essential for survival, but at the same time is also a major cause of human morbidity and mortality. 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. 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 model 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 CHRNA7 gene
The α7 nicotinic receptor is a homomeric receptor belonging to the ligand-gated ion channel receptor superfamily, composed of five α7 subunits, with a 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 of 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; at the post-synaptic level it is responsible for the fast excitatory post-synaptic potential.
The α7 receptor is also expressed in non-neuronal cells, such as endothelial cells, lymphocytes, keratinocytes, astrocytes, microglial cells and macrophages. In these not excitable cells the α7 receptor 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 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 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. 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. On the other hand, treatment of primary macrophages 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. Our hypothesis is that the CHRFAM7A gene product may be 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 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 anti-inflammatory 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 to identify the elements responsive to pro-inflammatory stimuli.
- the role of the CHRFAM7A isoforms in the regulation of CHRNA7 cellular localization.
Pubblicazioni di maggior rilievo
Fratangeli A, Parmigiani E, Fumagalli M, Lecca D, Benfante R, Passafaro M, Buffo A, Abbracchio MP, Rosa P. (2013). The regulated expression, intracellular trafficking, and membrane recycling of the P2Y-like receptor GPR17 in Oli-neu oligodendroglial cells. J Biol Chem. Feb 15;288(7):5241-56
Di Lascio S, Bachetti T, Saba E, Ceccherini I, Benfante R, Fornasari D. (2013). Transcriptional dysregulation and impairment of PHOX2B auto-regulatory mechanism induced by polyalanine expansion mutations associated with congenital central hypoventilation syndrome. Neurobiol Dis. Feb;50:187-200.
Benfante R, Antonini RA, De Pizzol M, Gotti C, Clementi F, Locati M, Fornasari D (2011). Expression of the α7 nAChR subunit duplicate form (CHRFAM7A) is down-regulated in the monocytic cell line THP-1 on treatment with LPS. J. Neuroimmunol. 230:74-84.
Benfante R, Antonini RA, Kuster N, Schuderer J, Maercker C, Adlkofer F, Clementi F, Fornasari D (2008). The expression of PHOX2A, PHOX2B and of their target gene dopamine-beta-hydroxylase (DbetaH) is not modified by exposure to extremely-low-frequency electromagnetic field (ELF-EMF) in a human neuronal model. Toxicol In Vitro. 22 (6):1489-1495.
Benfante R, Flora A, Di Lascio S, Cargnin F, Longhi R, Colombo S, Clementi F, Fornasari D. (2007). Transcription factor PHOX2A regulates the human alpha3 nicotinic receptor subunit gene promoter. J Biol Chem. 282(18):13290-302.
Antonini RA, Benfante R, Gotti C, Moretti M, Kuster N, Schuderer J, Clementi F, Fornasari D.(2006). Extremely low-frequency electromagnetic field (ELF-EMF) does not affect the expression of alpha3, alpha5 and alpha7 nicotinic receptor subunit genes in SH-SY5Y neuroblastoma cell line. Toxicol Lett. 164(3):268-77.
Solda G, Boi S, Duga S, Fornasari D, Benfante R, Malcovati M, Tenchini ML. (2005). In vivo RNA-RNA duplexes from human alpha3 and alpha5 nicotinic receptor subunit mRNAs. Gene. 345(2):155-64.
Benfante R, Antonini RA, Vaccari M, Flora A, Chen F, Clementi F, Fornasari D. (2005). The expression of the human neuronal alpha3 Na ,K -ATPase subunit gene is regulated by the activity of the Sp1 and NF-Y transcription factors. Biochem J. 386(Pt 1):63-72
Flora A, Schulz R, Benfante R, Battaglioli E, Terzano S, Clementi F, Fornasari D. (2000). Neuronal and extraneuronal expression and regulation of the human alpha5 nicotinic receptor subunit gene. J Neurochem. 2000 Jul;75(1):18-27.
Flora A, Schulz R, Benfante R, Battaglioli E, Terzano S, Clementi F, Fornasari D. (2000). Transcriptional regulation of the human alpha5 nicotinic receptor subunit gene in neuronal and non-neuronal tissues. Eur J Pharmacol. 2000 Mar 30;393(1-3):85-95.