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Small changes in bone structure of female ?7 nicotinic acetylcholine receptor knockout mice.

ABSTRACT: BACKGROUND:Recently, analysis of bone from knockout mice identified muscarinic acetylcholine receptor subtype M3 (mAChR M3) and nicotinic acetylcholine receptor (nAChR) subunit ?2 as positive regulator of bone mass accrual whereas of male mice deficient for ?7-nAChR (?7KO) did not reveal impact in regulation of bone remodeling. Since female sex hormones are involved in fair coordination of osteoblast bone formation and osteoclast bone degradation we assigned the current study to analyze bone strength, composition and microarchitecture of female ?7KO compared to their corresponding wild-type mice (?7WT). METHODS:Vertebrae and long bones of female 16-week-old ?7KO (n = 10) and ?7WT (n = 8) were extracted and analyzed by means of histological, radiological, biomechanical, cell- and molecular methods as well as time of flight secondary ion mass spectrometry (ToF-SIMS) and transmission electron microscopy (TEM). RESULTS:Bone of female ?7KO revealed a significant increase in bending stiffness (p < 0.05) and cortical thickness (p < 0.05) compared to ?7WT, whereas gene expression of osteoclast marker cathepsin K was declined. ToF-SIMS analysis detected a decrease in trabecular calcium content and an increase in C4H6N(+) (p < 0.05) and C4H8N(+) (p < 0.001) collagen fragments whereas a loss of osteoid was found by means of TEM. CONCLUSIONS:Our results on female ?7KO bone identified differences in bone strength and composition. In addition, we could demonstrate that ?7-nAChRs are involved in regulation of bone remodelling. In contrast to mAChR M3 and nAChR subunit ?2 the ?7-nAChR favours reduction of bone strength thereby showing similar effects as ?7?2-nAChR in male mice. nAChR are able to form heteropentameric receptors containing ?- and ?-subunits as well as the subunits ?7 can be arranged as homopentameric cation channel. The different effects of homopentameric and heteropentameric ?7-nAChR on bone need to be analysed in future studies as well as gender effects of cholinergic receptors on bone homeostasis.

SUBMITTER: Lips KS

PROVIDER: S-EPMC4328057 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Small changes in bone structure of female α7 nicotinic acetylcholine receptor knockout mice.

Lips Katrin S KS Yanko Özcan Ö Kneffel Mathias M Panzer Imke I Kauschke Vivien V Madzharova Maria M Henss Anja A Schmitz Peter P Rohnke Marcus M Bäuerle Tobias T Liu Yifei Y Kampschulte Marian M Langheinrich Alexander C AC Dürselen Lutz L Ignatius Anita A Heiss Christian C Schnettler Reinhard R Kilian Olaf O

BMC musculoskeletal disorders 20150131

<h4>Background</h4>Recently, analysis of bone from knockout mice identified muscarinic acetylcholine receptor subtype M3 (mAChR M3) and nicotinic acetylcholine receptor (nAChR) subunit α2 as positive regulator of bone mass accrual whereas of male mice deficient for α7-nAChR (α7KO) did not reveal impact in regulation of bone remodeling. Since female sex hormones are involved in fair coordination of osteoblast bone formation and osteoclast bone degradation we assigned the current study to analyze ...[more]

PMID: 25636336

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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.

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Small changes in bone structure of female ?7 nicotinic acetylcholine receptor knockout mice.

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Small changes in bone structure of female α7 nicotinic acetylcholine receptor knockout mice.

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