Dataset Information

Proteomic Investigation of Murine Neuronal ?7-Nicotinic Acetylcholine Receptor Interacting Proteins.

ABSTRACT: The ?7-nicotinic acetylcholine receptor (?7-nAChR) is a ligand-gated ion channel that is expressed widely in vertebrates and is the principal high-affinity ?-bungarotoxin (?-bgtx) binding protein in the mammalian CNS. ?7-nAChRs associate with proteins that can modulate its properties. The ?7-nAChR interactome is the summation of proteins interacting or associating with ?7-nAChRs in a protein complex. To identify an ?7-nAChR interactome in neural tissue, we isolated ?-bgtx-affinity protein complexes from wild-type and ?7-nAChR knockout (?7 KO) mouse whole brain tissue homogenates using ?-bgtx-affinity beads. Affinity precipitated proteins were trypsinized and analyzed with an Orbitrap Fusion mass spectrometer. Proteins isolated with the ?7-nAChR specific ligand, ?-bgtx, were determined to be ?7-nAChR associated proteins. The ?7-nAChR subunit and 120 additional proteins were identified. Additionally, 369 proteins were identified as binding to ?-bgtx in the absence of ?7-nAChR expression, thereby identifying nonspecific proteins for ?7-nAChR investigations using ?-bgtx enrichment. These results expand on our previous investigations of ?7-nAChR interacting proteins using ?-bgtx-affinity bead isolation by controlling for differences between ?7-nAChR and ?-bgtx-specific proteins, developing an improved protein isolation methodology, and incorporating the latest technology in mass spectrometry. The ?7-nAChR interactome identified in this study includes proteins associated with the expression, localization, function, or modulation of ?7-nAChRs, and it provides a foundation for future studies to elucidate how these interactions contribute to human disease.

SUBMITTER: Mulcahy MJ

PROVIDER: S-EPMC6301012 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Proteomic Investigation of Murine Neuronal α7-Nicotinic Acetylcholine Receptor Interacting Proteins.

Mulcahy Matthew J MJ Paulo Joao A JA Hawrot Edward E

Journal of proteome research 20181004 11

The α7-nicotinic acetylcholine receptor (α7-nAChR) is a ligand-gated ion channel that is expressed widely in vertebrates and is the principal high-affinity α-bungarotoxin (α-bgtx) binding protein in the mammalian CNS. α7-nAChRs associate with proteins that can modulate its properties. The α7-nAChR interactome is the summation of proteins interacting or associating with α7-nAChRs in a protein complex. To identify an α7-nAChR interactome in neural tissue, we isolated α-bgtx-affinity protein comple ...[more]

PMID: 30285449

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Project description:PurposeThe α7 nicotinic acetylcholine receptor (nAChR) is widely expressed in the nervous system, including in the inner retinal neurons in all species studied to date. Although reductions in the expression of α7 nAChRs are thought to contribute to the memory and visual deficits reported in Alzheimer's disease (AD) and schizophrenia , the α7 nAChR knockout (KO) mouse is viable and has only slight visual dysfunction. The absence of a major phenotypic abnormality may be attributable to developmental mechanisms that serve to compensate for α7 nAChR loss. We hypothesized that the upregulation of genes encoding other nAChR subunits or muscarinic acetylcholine receptor (mAChR) subtypes during development partially accounts for the absence of major deficiencies in the α7 nAChR KO mouse. The purpose of this study was to determine whether the deletion of the α7 nAChR subunit in a mouse model resulted in changes in the regulation of other cholinergic receptors or other ion channels in an α7 nAChR KO mouse when compared to a wild-type (WT) mouse.MethodsTo examine gene expression changes, we employed a quantitative real-time polymerase chain reaction (qPCR) using whole retina RNA extracts as well as RNA extracted from selected regions of the retina. These extracts were collected using laser capture microdissection (LCM). The presence of acetylcholine receptor (AChR) subunit and subtype proteins was determined via western blotting. To determine any differences in the number and distribution of choline acetyltransferase (ChAT) amacrine cells, we employed wholemount and vertical immunohistochemistry (IHC) and cell counting. Additionally, in both WT and α7 nAChR KO mouse retinas, the distribution of the nAChR subunit and mAChR subtype proteins were determined via IHC for those KO mice that experienced mRNA changes.ResultsIn the whole retina, there was a statistically significant upregulation of α2, α9, α10, β4, nAChR subunit, and m1 and m4 mAChR subtype transcripts in the α7 nAChR KO mice. However, the retinal layers showed complex patterns of transcript expression. In the ganglion cell layer (GCL), m2 and m4 mAChR subtype transcripts were significantly upregulated, while β3 and β4 nAChR subunit transcripts were significantly downregulated. In the inner portion of the inner nuclear layer (iINL), α2, α9, β4, nAChR subunit, and m3 and m4 mAChR subtype transcripts were significantly downregulated. In the outer portion of the inner nuclear layer (oINL), β2, β4, and m4 AChR subunit transcripts were significantly upregulated. Western blot experiments confirmed the protein expression of α3-α5 and α9-containing nAChR subunits and m1-m2 mAChR subtypes in mouse retinas. IHC results supported many of the mRNA changes observed. Finally, this is the first report of α9 and α10 nAChR subunit expressions in the retina of any species.ConclusionsRather than a simple upregulation of a single AChR subunit or subtype, the absence of the α7 nAChR in the KO mice was associated with complex layer-specific changes in the expression of AChR subunits and subtypes.

Project description:BackgroundWomen with endometriosis have been shown to have a reduced vagal tone as compared with controls and vagotomy promoted while vagus nerve stimulation (VNS) decelerated the progression of endometriosis in mice. Extensive research also has shown that the activation of the cholinergic anti-inflammatory pathway by VNS activates α7 nicotinic acetylcholine receptor (α7nAChR), potently reducing inflammation. Yet whether α7nAChR plays any role in endometriosis is unknown. We evaluated its expression in normal endometrium, ovarian and deep endometriotic lesions, and evaluated its role in the development of endometriosis.MethodsImmunohistochemistry analyses of α7nAChR in endometriotic lesions as well as control endometrium, and quantification of tissue fibrosis by Masson trichrome staining were performed. Mouse experiments were conducted to evaluate the impact of α7nAChR activation or suppression on lesional progression and possible therapeutic effect. Finally, in vitro experiments were conducted to evaluate the effect of activation of α7nAChR on epithelial-mesenchymal transition (EMT), fibroblast-to-myofibroblast transdifferentiation (FMT), smooth muscle metaplasia (SMM) and fibrogenesis in an endometriotic epithelial cell line and primary endometriotic stromal cells derived from ovarian endometrioma tissue samples.ResultsImmunostaining of α7nAChR was significantly reduced in human endometriotic epithelial cells as compared with their counterpart in normal endometrium. Lesional α7nAChR staining levels correlated negatively with lesional fibrosis and the severity of dysmenorrhea. The α7nAChR agonist significantly impeded the development of endometriotic lesions in mouse models possibly through hindrance of EMT and FMT. It also demonstrated therapeutic effects in mice with induced deep endometriosis. Treatment of endometriotic epithelial and stromal cells with an α7nAChR agonist significantly abrogated platelet-induced EMT, FMT and SMM, and suppressed cellular contractility and collagen production.Conclusionsα7nAChR is suppressed in endometriotic lesions, and its activation by pharmacological means can impede EMT, FMT, SMM, and fibrogenesis of endometriotic lesions. As such, α7nAChR can be rightfully viewed as a potential target for therapeutic invention.Trial registrationNot applicable.

Dataset Information

Proteomic Investigation of Murine Neuronal ?7-Nicotinic Acetylcholine Receptor Interacting Proteins.

Publications

Proteomic Investigation of Murine Neuronal α7-Nicotinic Acetylcholine Receptor Interacting Proteins.

Similar Datasets

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