Accumulation of the age pigment lipofuscin represents a ubiquitous hallmark of the aging process. However, our knowledge about cellular effects of lipofuscin accumulation is potentially flawed, because previous research mainly utilized highly artificial methods of lipofuscin generation. In order to address this tremendous problem, we developed a convenient protocol for isolation of authentic lipofuscin from human and equine cardiac tissue in high purity and quantity. Isolated lipofuscin aggregates contained elevated concentrations of proline and metals such as calcium or iron. The material was readily incorporated by fibroblasts and caused cell death at low concentrations (LC₅₀ = 5.0 μg/mL) via a pyroptosis-like pathway. Lipofuscin boosted mitochondrial ROS production and caused lysosomal dysfunction by lysosomal membrane permeabilization leading to reduced lysosome quantity and impaired cathepsin D activity. In conclusion, this is the first study utilizing authentic lipofuscin to experimentally validate the concept of the lysosomal-mitochondrial axis theory of aging and cell death.

Enzymatic and non-enzymatic posttranslational protein modifications by oxidation, glycation and acylation are key regulatory mechanisms in hallmarks of aging like inflammation, altered epigenetics and decline in proteostasis. In this study a mouse cohort was used to monitor changes of posttranslational modifications in the aging process. A protocol for the extraction of histones, cytosolic and mitochondrial proteins from mouse liver was developed and validated. In total, 6 lysine acylation structures, 7 advanced glycation endproducts, 6 oxidative stress markers, and citrullination were quantitated in proteins of subcellular compartments using HPLC-MS/MS. Methionine sulfoxide, acetylation, formylation, and citrullination were the most abundant modifications. Histone proteins were extraordinary high modified and non-enzymatic modifications accumulated in all subcellular compartments during the aging process. Compared to acetylation of histone proteins which gave between 350 and 305 µmol/mol leucine equivalents in young and old animals, modifications like acylation, glycation, and citrullination raised to 43%, 20%, and 18% of acetylation, respectively. On the other hand there was an age related increase of selected oxidative stress markers by up to 150%. The data and patterns measured in this study are mandatory for further studies and will strongly facilitate understanding of the molecular mechanisms in aging.

Protein glycation is usually referred to as an array of non-enzymatic post-translational modifications formed by reducing sugars and carbonyl products of their degradation. The resulting advanced glycation end products (AGEs) represent a heterogeneous group of covalent adducts, known for their pro-inflammatory effects in mammals, and impacting on pathogenesis of metabolic diseases and ageing. In plants, AGEs are the markers of tissue ageing and response to environmental stressors, the most prominent of which is drought. Although water deficit enhances protein glycation in leaves, its effect on seed glycation profiles is still unknown. Moreover, the effect of drought on biological activities of seed protein in mammalian systems is still unstudied with respect to glycation. Therefore, here we address the effects of a short-term drought on the patterns of seed protein-bound AGEs and accompanying alterations in pro-inflammatory properties of seed protein in the context of seed metabolome dynamics. A short-term drought, simulated as polyethylene glycol-induced osmotic stress and applied at the stage of seed filling, resulted in the dramatic suppression of primary seed metabolism, although the secondary metabolome was minimally affected. This was accompanied with significant suppression of NF-kB activation in human SH-SY5Y neuroblastoma cells after a treatment with protein hydrolyzates, isolated from the mature seeds of drought-treated plants. This effect could not be attributed to formation of known AGEs. Most likely, the prospective anti-inflammatory effect of short-term drought is related to antioxidant effect of unknown secondary metabolite protein adducts, or down-regulation of unknown plant-specific AGEs due to suppression of energy metabolism during seed filling.

Aging represents the accumulation of changes in an individual over time, encompassing physical, psychological, and social changes. Posttranslational modifications of proteins such as glycosylation, including sialylation or glycation, are proposed to be involved in this process, since they modulate a variety of molecular and cellular functions. In this study, we analyzed selected posttranslational modifications and the respective proteins on which they occur in young and old mouse brains. The expression of neural cell adhesion molecule (NCAM), receptor for advanced glycation endproducts (RAGE), as well as the carbohydrate-epitopes paucimannose and high-mannose, polysialic acid, and O-GlcNAc were examined. We demonstrated that mannose-containing glycans increased on glycoproteins in aged mouse brains and identified synapsin-1 as one major carrier of paucimannose in aged brains. In addition, we found an accumulation of so-called advanced glycation endproducts, which are generated by non-enzymatic reactions and interfere with protein function. Furthermore, we analyzed the expression of sialic acid and found also an increase during aging.