Exploring a reliable and sensitive method to detect telomere dysfunction in the aging population and premature-aging diseases

National Institute On Aging

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Abstract

Telomere dysfunction-induced foci (TIF) are detected in senescent human fibroblasts and aged murine and primate tissues and have recently been used as a marker for aging. Currently, TIF is determined by the colocalization of telomeric DNA/protein and phosphorylated histone H2AX (gamma-H2AX) using indirect immunofluorescence or indirect immunofluorescence combined with telomere FISH. However, due to the incapability of these conventional assays to detect TIFs at short telomeres, it is unclear if eroded telomeres in human conditions, such as aging and age-related diseases become dysfunctional, thereby contributing to the DNA damage signaling and cellular senescence. We are developing a novel method, employing proximity ligation assay (PLA), to detect, visualize, and quantify TIFs at single-cell resolution. Comparing to conventional methods, PLA significantly increases detection sensitivity up to 1000 times and eliminates false positive and non-specific signals. Using this method, we have measured TIFs in primary fibroblasts derived from patients with short telomere syndrome, dyskeratosis congenita (DC) and from human healthy population at various ages. We observed that DC fibroblasts displayed TIF foci accumulation. In addition, older individuals did not show significant TIF formation, compared to younger individuals. These studies validate the sensitivity and reliability of our PLA assay. Our data will enhance our understanding of the role of telomere attrition in human aging and diseases. We will continue to explore PLA assay conditions in human peripheral blood mononuclear cells.