CARATTERIZZAZIONE MOLECOLARE E FUNZIONALE DEL GENE CHRFAM7A, FORMA DUPLICATA DELLA SUBUNITÀ ALPHA7 DEL RECETTORE NICOTINICO.

The α7 nicotinic acetylcholine receptor (α7 nAChR) has a key role in the innate immune system’s inflammatory response, as part of “cholinergic anti-inflammatory pathway”: a process by which acetylcholine from the vagus nerve reduces the release of the pro-inflammatory cytokine TNFα, thus allowing for a controlled response to infection. The CHRNA7 gene, in humans, is partially duplicated from exon 5-10 and forms an hybrid with four exons (D-A) of a novel gene, FAM7A. This new gene, CHRFAM7A, which is located in the opposite orientation, at 1.6 Mb, from CHRNA7, is not present on every chromosome 15 and a polymorphic variant, in linkage disequilibrium with a 2bp deletion in exon 6, in the same orientation to the CHRNA7 gene, has been described in a cohort of patients with bipolar disorders and schizophrenia. THP-1 monocytic-like cell line expresses only CHRFAM7A, which was down-regulated on treatment with LPS, by a direct transcriptional mechanism reliant on NF-kB. This effect has been confirmed in primary monocytes and macrophages cell cultures, where CHRFAM7A is expressed 200-1000 times more than CHRNA7. Here, the conventional α7 subunit was up-regulated by LPS treatment, thus suggesting the involvement of CHRFAM7A in the regulation of cell surface α7 receptors’ level (a mechanism unique to humans) and the ability of immune cells to respond to acetylcholine, released from the vagus nerve, during infection. This hypothesis seems to be supported by recent works showing that the duplicated form may have a dominant negative effect on the activity of α7 nAChR. Infact, co-expression of CHRFAM7A with α7 results in a significant reduction of the Ach-evoked currents, suggesting the presence of heteromeric non functional receptors at the plasma membrane. The promoter region that regulates the expression of CHRFAM7A is still unknown. To try to identify and characterize this region, 5'-RACE experiments were carried out to map the CHRFAM7A mRNA 5’UTR. RNA was extracted from three different cell lines: THP-1 cells, primary human macrophages and SHSY5Y neuroblastoma cell line. Multiple transcription start sites were identified, depending on the cell line used, suggesting the existence of alternative promoters. A series of constructs that recapitulate the mapping of the CHRFAM7A regulatory region, according to the transcription start sites identified, was also generated. They were cloned into a reporter vector and their functionality was tested by transient transfection both in THP-1 and SHSY5Y cell models. Through these experiments, an intronic region (-702/-208 bp from ATG codon, in exon B) and an Alu sequence (-1155/-821 bp) were identified as negative regulators of reporter gene transcription. Future experiments will allow us to identify other regulatory sites, important for proper CHRFAM7A gene expression in different tissues. Furthermore, two variants exist for CHRFAM7A gene, due to alternative splicing, that gives rise to two protein products of predicted 36 and 47 KDa, whose function is currently unknown. The N-terminally portion of each variant would lack the majority of the ligand binding domain, but the protein product retains the transmembrane region that forms the ion channel. To clarify the function of CHRFAM7A gene and to study the cellular localization of these two isoforms, CHRFAM7A (both variants) and CHRNA7 tagged proteins were generated, by cloning their cDNAs into pcDNA3.1/myc-His and pcDNA3.1/V5-His vectors. The α7 specific chaperon, RIC3, was also cloned into pcDNA3.0 vector and co-transfected with the tagged proteins, in order to increase their folding and expression. The constructs were transfected into Hela cells and characterized by immunofluorescence and western blotting experiments. The use of nicotine as therapy for chronic inflammatory diseases has often been characterised by excessive side effects due to a lack of specificity for just one receptor type. For this reason, the study of the regulation of α7 and its duplicated isoform subunits in response to pro-inflammatory stimuli, is important to understand their role in the “cholinergic anti-inflammatory pathway”, in order to discover selective nicotinic receptor agonists and to develop novel anti-inflammatory treatments.