Expression of RAD21 immunoreactivity in myenteric neurons of the human and mouse small intestine

Background RAD21 is a double‐strand‐break repair protein and component of the cohesin complex with key roles in cellular functions. A RAD21 loss‐of‐function mutation was found in cases of chronic intestinal pseudo‐obstruction (CIPO) with associated enteric neuronal loss. Analysis of RAD21 expression...

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Published in:	Neurogastroenterology and motility Vol. 30; no. 9; pp. e13429 - n/a
Main Authors:	Bianco, F., Eisenman, S. T., Colmenares Aguilar, M. G., Bonora, E., Clavenzani, P., Linden, D. R., De Giorgio, R., Farrugia, G., Gibbons, S. J.
Format:	Journal Article
Language:	English
Published:	England Wiley Subscription Services, Inc 01-09-2018
Subjects:	Cell culture Choline chronic intestinal pseudo‐obstruction Cohesin Enteric nervous system enteric neurons Ganglia Glial fibrillary acidic protein Immunofluorescence Immunoreactivity Interstitial cells Interstitial cells of Cajal Jejunum Mutation Myenteric plexus Neonates Neuronal-glial interactions Neurons Neuropeptide Y Nitric oxide Nitric-oxide synthase Rodents Small intestine enteric neurons chronic intestinal pseudo-obstruction immunofluorescence
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Summary:	Background RAD21 is a double‐strand‐break repair protein and component of the cohesin complex with key roles in cellular functions. A RAD21 loss‐of‐function mutation was found in cases of chronic intestinal pseudo‐obstruction (CIPO) with associated enteric neuronal loss. Analysis of RAD21 expression in the enteric nervous system is lacking, thus we aimed to characterize RAD21 immunoreactivity (IR) in myenteric ganglia. Methods Double labeling immunofluorescence in mouse and human jejunum was used to determine colocalization of RAD21 with HuC/D, PGP9.5, neuronal nitric oxide synthase (nNOS), neuropeptide Y (NPY), choline acetyl transferase (ChAT), Kit, platelet‐derived growth factor receptor‐α (PDGFRα), and glial fibrillary acid protein (GFAP) IRs. Results A subset of PGP9.5‐ and HuC/D‐IR neuronal cell bodies and nerve fibers in the myenteric plexus of human and mouse small intestine also displayed cytoplasmic RAD21‐IR Cytoplasmic RAD21‐IR was found in 43% of HuC/D‐IR neurons in adult and neonatal mice but did not colocalize with nNOS. A subset of ChAT‐positive neurons had cytoplasmic RAD21‐IR Punctate RAD21‐IR was restricted to the nucleus in most cell types consistent with labeling of the cohesin complex. Cytoplasmic RAD21‐IR was not detected in interstitial cells of Cajal, fibroblast‐like cells or glia. Subsets of neurons in primary culture exhibited cytoplasmic RAD21‐IR Suppression of RAD21 expression by shRNA knockdown abolished RAD21‐IR in cultured neurons. Conclusions Our data showing cytoplasmic RAD21 expression in enteric neurons provide a basis toward understanding how mutations of this gene may contribute to altered neuronal function/survival thus leading to gut‐motor abnormalities. RAD21 is a double‐strand‐break repair protein and a component of the cohesin complex. A RAD21 loss‐of‐function mutation was found in cases of chronic intestinal pseudo‐obstruction with associated enteric neuronal loss. We found RAD21 immunolabeling in the cytoplasm of cholinergic but not nitrergic myenteric neurons, consistent with differential roles of RAD21 in neuronal function/survival and possibly gut‐motor abnormalities.
Bibliography:	Funding information This work was supported by grants from University of Bologna (RFO funds) (to RDeG), the Telethon Foundation (GGP15171) of Italy (to RDeG and EB) and the ‘Fondazione del Monte di Bologna e Ravenna’, Bologna, Italy (to RDeG) and the NeuroGut Initial Training Network (607652 – ITN) part of the FP7 from the European Community (RDeG). US National Institutes of Health, P30DK084567, R01DK57061, and R01DK106011. Funding bodies did not influence the content of this article. ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	1350-1925 1365-2982
DOI:	10.1111/nmo.13429