Evaluation of GMI ganglioside-mediated apoptosis in feline thymocytes

Abstract

Cats with inherited GM1 gangliosidosis (GM1 mutant cats) have premature thymic involution characterized by decreased total thymocytes primarily affecting the CD4 + CD8 + subpopulation. While GM1 mutant cats have increased cell surface GM1 gangliosides, as determined by cholera toxin B binding, on both thymocytes and peripheral lymph node cells only thymocytes show increased apoptosis. To determine if GM1 gangliosides can increase the occurrence of apoptosis in feline thymocytes directly, we added exogenous GM1 ganglioside (GM1) to feline thymocyte primary cultures and compared the results to apoptotic changes seen in untreated cells or in cells treated with dexamethasone (Dex), a known inducer of thymocyte apoptosis in other species. Incorporation of exogenous GM1 into thymocyte cytoplasmic membranes was confirmed by flow cytometric analyses of cholera toxin B labelling. Apoptosis in feline thymocytes was analyzed by electron microscopy, spectrophotometric evaluation of DNA fragmentation, flow cytometric enumeration of apoptotic nuclei, and gel electrophoretic analysis of degraded DNA. Alterations in percentages of thymocyte immunophenotype following GM1 incorporation were determined by flow cytometric analyses of labelled cell surface markers for feline CD4 and CD8. Because in vitro addition of GM1 gangliosides has been reported in other species to decrease surface expression of CD4 on both thymocytes and peripheral lymphocytes, we evaluated GM1-associated downregulation of CD4 on the surface of feline thymocytes and peripheral lymph node cells by flow cytometry. Additionally, we compared the apoptotic response of the more mature peripheral lymph node cells to the less mature thymocytes. Our results indicate that incorporation of exogenous GM1 into feline thymocyte cell membranes produces a dose-dependent increase of apoptotic cell death. Although, CD4 expression on both feline thymocyte and lymph node cell membranes was abruptly decreased after introducing exogenous GM1, enhanced apoptotic death was observed only in thymocytes, not in lymph node cells at the same GM1 concentration. Enhancement of thymocyte apoptosis appears to be age-related since cells derived from cats <3 months of age were more vulnerable than those from cats >3 months of age.