Andrea Lorincz

Ano1 is a selective marker of interstitial cells of Cajal in the human and mouse gastrointestinal tract

Populations of interstitial cells of Cajal (ICC) are altered in several gastrointestinal neuromuscular disorders. ICC are identified typically by ultrastructure and expression of Kit (CD117), a protein that is also expressed on mast cells. No other molecular marker currently exists to independently identify ICC. The expression of ANO1 (DOG1, TMEM16A), a Ca(2+)-activated Cl(-) channel, in gastrointestinal stromal tumors suggests it may be useful as an ICC marker. The aims of this study were therefore to determine the distribution of Ano1 immunoreactivity compared with Kit and to establish whether Ano1 is a reliable marker for human and mouse ICC. Expression of Ano1 in human and mouse stomach, small intestine, and colon was investigated by immunofluorescence labeling using antibodies to Ano1 alone and in combination with antibodies to Kit. Colocalization of immunoreactivity was demonstrated by epifluorescence and confocal microscopy. In the muscularis propria, Ano1 immunoreactivity was restricted to cells with the morphology and distribution of ICC. All Ano1-positive cells in the muscularis propria were also Kit positive. Kit-expressing mast cells were not Ano1 positive. Some non-ICC in the mucosa and submucosa of human tissues were Ano1 positive but Kit negative. A few (3.2%) Ano1-positive cells in the human gastric muscularis propria were labeled weakly for Kit. Ano1 labels all classes of ICC and represents a highly specific marker for studying the distribution of ICC in mouse and human tissues with an advantage over Kit since it does not label mast cells.

Loss of Kitlow progenitors, reduced stem cell factor and high oxidative stress underlie gastric dysfunction in progeric mice

Gastrointestinal functions decline with ageing leading to impaired quality of life, and increased morbidity and mortality. Neurodegeneration is believed to underlie ageing‐associated dysmotilities but the mechanisms have not been fully elucidated. We used progeric mice deficient in the anti‐ageing peptide Klotho to investigate the contribution of key cell types of the gastric musculature to ageing‐associated changes in stomach function and the underlying mechanisms. Klotho expression, enteric neurons, interstitial cells of Cajal (ICC), smooth muscle cells and electrical activity were assessed by immunofluorescence, confocal microscopy, 3‐dimensional reconstruction, flow cytometry, quantitative RT‐PCR, Western immunoblotting and intracellular recordings. Gastric emptying of solids was analysed by the [13C]octanoic acid breath test. Circulating and tissue trophic factors were measured by enzyme immunoassays and quantitative RT‐PCR. The role of oxidative stress was investigated in organotypic cultures. Klotho expression was detected in gastric glands, myenteric neurons and smooth muscle cells. Progeric Klotho‐deficient mice had profound loss of ICC and ICC stem cells without a significant decrease in neuron counts, expression of neuronal nitric oxide synthase or smooth muscle myosin. Slow wave amplitude and nitrergic inhibitory junction potentials were reduced while solid emptying was unchanged. Klotho‐deficient mice were marantic and had low insulin, insulin‐like growth factor‐I and membrane‐bound stem cell factor. Klotho deficiency accentuated oxidative stress and ICC loss. We conclude that Klotho‐deficient, progeric mice display a gastric phenotype resembling human ageing and involving profound ICC loss. Klotho protects ICC by preserving their precursors, limiting oxidative stress, and maintaining nutritional status and normal levels of trophic factors important for ICC differentiation.

Generalized neuromuscular hypoplasia, reduced smooth muscle myosin and altered gut motility in the klotho model of premature aging.

Background  Gastrointestinal symptoms, particularly constipation, increase with aging, but their underlying mechanisms are poorly understood due to lack of experimental models. Previously we established the progeric klotho mouse as a model of aging‐associated anorexia and gastric dysmotility. We also detected reduced fecal output in these animals; therefore, the aim of this study was to investigate in vivo function and cellular make‐up of the small intestinal and colonic neuromuscular apparatus.