UNIVERSITA’ DEGLI STUDI DI MILANO Dipartimento di Biotecnologie Mediche e Medicina Traslazionale Scuola di dottorato in Medicina Sperimentale e Biotecnologie Mediche Ciclo XXIX THE HUMAN-RESTRICTED DUPLICATED FORM OF THE α7 NICOTINIC ACETYLCHOLINE RECEPTOR, CHRFAM7A: TRANSCRIPTIONAL REGULATION, ROLE IN INFLAMMATION AND POTENTIAL PHARMACOLOGICAL IMPLICATIONS. Settore disciplinare: Bio/14 Tesi di dottorato di: Annalisa Maroli Relatore: Prof.ssa Grazia Pietrini Coordinatore: Prof. Massimo Locati Anno accademico: 2016-2017 1 2 Contents Abstract ................................................................................................................................. 5 1. Introduction ................................................................................................................... 7 1.1. Inflammation........................................................................................................... 7 1.1.1. Acute inflammation ......................................................................................... 7 1.1.2. Molecular mechanisms of acute inflammation ............................................... 9 1.1.3. Resolution of acute inflammation ................................................................. 13 1.1.4. Chronic inflammation .................................................................................... 14 1.2. The Cholinergic Anti-Inflammatory Pathway........................................................ 14 1.3. The α7 nicotinic acetylcholine receptor ............................................................... 18 1.3.1. The CHRNA7 gene .......................................................................................... 21 1.3.2. α7 nicotinic acetylcholine protein in the nervous system ............................ 23 1.3.3. Function of the α7 nicotinic acetylcholine receptor in the immune system 24 1.3.4. α7nAChR-related pathologies ....................................................................... 26 1.3.5. α7nAChR involvement in AD therapy: the case of Donepezil ....................... 31 1.4. The CHRFAM7A gene ............................................................................................ 33 1.4.1. The CHRFAM7A locus .................................................................................... 33 1.4.2. The CHRFAM7A transcript ............................................................................. 35 1.4.3. The α7dup protein ......................................................................................... 38 1.4.4. CHRFAM7A involvement in human disease .................................................. 44 2. Aim of the project ........................................................................................................ 48 3. Materials and Methods ............................................................................................... 50 3.1. Cell Lines ............................................................................................................... 50 3.2. Cell treatment ....................................................................................................... 50 3.3. 5’ RACE .................................................................................................................. 51 3.4. Total RNA Extraction and Reverse Transcription .................................................. 52 3.5. Standard PCR protocol using GoTaq Flexi (Promega) ........................................... 52 3.6. High Fidelity PCR using Expand High Fidelity PCR System (Roche) ...................... 53 3.7. Quantitative Real-Time PCR .................................................................................. 53 3.8. Absolute quantification by Real-Time RT-PCR ...................................................... 54 3.9. Plasmid vectors ..................................................................................................... 55 3 3.10. Transient transfection and luciferase assay ...................................................... 60 3.11. Data analysis ...................................................................................................... 60 3.12. Chromatin Immunoprecipitation ...................................................................... 61 3.13. Electrophoresis Mobility Shift Assay ................................................................. 62 3.14. Human biological samples ................................................................................ 63 4. Results.......................................................................................................................... 65 4.1. Characterization of CHRFAM7A transcriptional regulation .................................. 65 4.1.1. Identification of the CHRFAM7A regulatory region ...................................... 65 4.1.2. Identification of multiple Transcription Start Sites (TSS) in monocytic cell line, primary human macrophages and neuroblastoma cell model ........................... 67 4.1.3. Functional analysis of the CHRFAM7A regulatory sequence in THP-1 and SH- SY5Y cell line ................................................................................................................ 73 4.1.4. The CHRFAM7A Intron 4 reduces the transcriptional activity in THP-1 but not in SH-SY5Y cell model .................................................................................................. 77 4.1.5. Identification of a novel CHRFAM7A Transcription Start Site in intron 5 ..... 81 4.1.6. LPS treatment causes chromatin remodelling at the CHRFAM7A promoter 84 4.1.7. LPS treatment decreases CHRFAM7A promoter activity .............................. 87 4.1.8. Summary ........................................................................................................ 90 4.2. CHRFAM7A as pharmacological target in Alzheimer’s disease: a report of Donepezil effect ............................................................................................................... 90 4.2.1. CHRNA7 and CHRFAM7A expression levels in hippocampus of AD patients 90 4.2.2. CHRNA7 and CHRFAM7A expression level in human PBMCs of AD untreated and Donepezil treated patients ................................................................................... 93 4.2.3. Donepezil up-regulates CHRFAM7A transcript in THP-1 cell model ............. 94 4.2.4. Donepezil treatment on human primary macrophages modulates CHRNA7 and CHRFAM7A transcript ......................................................................................... 102 4.2.5. Donepezil treatment up-regulates CHRFAM7A and down-regulates CHRNA7 gene in SH-SY5Y cell model ....................................................................................... 105 4.2.6. Summary ...................................................................................................... 106 5. Discussion .................................................................................................................. 108 6. Conclusions ................................................................................................................ 121 Acknowledgment ............................................................................................................... 123 Bibliography ....................................................................................................................... 124 4 Abstract The α7 nicotine acetylcholine receptor (α7nAChR, CHRNA7) is a homo-pentameric ligand-gated ion channel widely expressed in the Central Nervous System (CNS). Recent evidence has demonstrated its expression also in non-neuronal tissues, including monocytes and macrophages, where it mediates the Cholinergic Anti-Inflammatory Pathway, a neuronal reflex providing the central control of systemic inflammation. Recently, the human-restricted duplicated gene of the α7nAChR, called CHRFAM7A (α7dup) has been discovered. It is the product of the partial duplication and fusion of exon 5-10 of CHRNA7 gene with the novel exons D, C, B and A belonging to the FAM7A gene, of unknown function. The CHRFAM7A gene is expressed in human immune cells and in the CNS and is translated into two proteins, of 45 kDa and 36 kDa, which are the result of alternative splicing. The α7dup protein assembles with the α7 conventional subunits and exerts a dominant negative regulation on the α7nAChR function. The importance of the α7dup protein in the human inflammatory process has been confirmed by the demonstration of its responsiveness to pro-inflammatory stimuli: indeed, the Lipopolysaccharide (LPS) treatment of THP-1 monocytic cells and of human primary monocytes and macrophages down-regulates CHRFAM7A transcript and protein through a transcriptional mechanism reliant on the NF-κB transcription factor. Moreover, unpublished data demonstrated that LPS has the opposite effect on the α7 transcript in monocytes and macrophages, leading to CHRNA7 up-regulation. In this study, we have investigated the transcriptional mechanisms leading to CHRFAM7A expression in the THP-1 monocytic cells and in neuroblastoma SH-SY5Y cells and we demonstrate that the CHRFAM7A gene is endowed with several complex transcriptional mechanisms leading to fine expression modulation, including the presence of alternative and tissue-specific Transcription Start Site (TSS), alternative splicing mechanism, tissue-specific transcriptional elements and intronic silencer elements. Moreover, we demonstrated that the CHRFAM7A down-regulation exerted by LPS involve the chromatin remodeling