An Improved System for Testing Lipofuscin Removal

A team funded by the SENS Research Foundation has developed an improved system for evaluating methods of lipofuscin removal in cells. Lipofuscin, a mix of many different forms of hardy metabolic waste, builds up in tissues with age. It clogs up lysosomes, the cellular recycling units, causing them to become bloated and dysfunctional. As all forms of cellular garbage accumulate due to this issue, cells themselves become dysfunctional or die. This is a significant and damaging problem in long-lived cell populations, such as those of the central nervous system. The compounds involved in the lipofuscin mix found in the retina, for example, directly contribute to the progressive age-related blindness caused by macular degeneration. Elsewhere in the body lipofuscin accumulation is implicated in the pathology of a range of age-related diseases. Clearing these waste compounds is an important goal in the development of rejuvenation therapies to treat aging by addressing its root causes.

Lipofuscin accumulation has an inverse relationship with lifespan and is a well-documented hallmark of aging. Many age-related disease states including Alzheimer's, Parkinson's, and age-related macular degeneration show increased lipofuscin accumulation. Some organisms accumulate lipofuscin in a nearly linear manner over time, and therefore their age is determined using methods that quantify lipofuscin levels. Two primary theories have been proposed for lipofuscin formation: the mitochondrial-lysosomal axis theory of aging and the protease inhibitor model of aging. The former focuses on irreparable oxidative damage caused by oxygen-driven Fenton reactions associated with mitochondrial processes, while the latter espouses inadequate lysosomal proteolysis as a cause of aging. Both theories have significant merit and lend credence to the 'garbage catastrophe' theory of aging, which states that the buildup of recalcitrant nondegradable material within the cell eventually leads to cell senescence or inhibited function.

Since lipofuscin accumulation impairs proteosome and lysosome pathways critical to cell health and homeostasis, the ability to quickly generate lipofuscin in vitro, and identify drugs that mitigate the accumulation or clear lipofuscin would be of great benefit to aging research. Here, we present a platform to quickly create lipofuscin-loaded but otherwise healthy cells and screen drugs for efficacy in lipofuscin removal. The combination of leupeptin, iron (III) chloride and hydrogen peroxide generates significant amounts of lipofuscin within cells while eliminating the need for a 40% hyperoxic chamber required by another existing protocol for lipofuscin generation. Alternative methods which load fibroblasts with "artificial" lipofuscin obtained via UV-peroxidation of mitochondrial fragments are much more labor-intensive. This method is faster (≤10 days) than most protocols to generate lipofuscin and assess its removal, which typically require 2 to 4 weeks or longer to complete.



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