Failing Autophagy and Lipofuscin Accumulation in the Aging Brain
It is known that the cellular housekeeping process of autophagy declines with aging, and it is also known that the metabolic waste known as lipofuscin builds up in long-lived cells at the same time. In the SENS view of aging, this lipofuscin accumulation is one of the causes of failing autophagy, as it accumulates in the recycling structures called lysosomes, degrading their function. Definitively proving this direction of causation, versus it being the other way around, is ever a challenge, however. The most effective way to do that is to clear out lipofuscin in old tissues and then observe the results, but at present this can only be achieved for a few of the many constituent compounds that make up this form of waste.
Autophagy is a self-degradative, highly regulated process that involves the non-specific degradation of cytoplasmic macromolecules and organelles via the lysosomal system. There are three different autophagic pathways based on the mechanisms for delivery of cargo to lysosomes: macroautophagy, microautophagy and chaperone-mediated autophagy (CMA). Macroautophagy (herein referred to as autophagy) is the major lysosomal pathway for the turnover of cytoplasmic components. Emerging evidence indicates that autophagy protects cells by removing long-lived proteins, aggregated protein complexes, and excess or damaged organelles. Defects in autophagy, therefore, are associated to various pathological conditions within organisms, including tumorigenesis, defects in developmental programs and the build-up of toxic, protein aggregates involved in neurodegeneration such as Amyloid precursor protein (APP). It has been recently suggested that the progressive age-related decline of autophagic and lysosomal activity may also be responsible for the continuous intraneuronal accumulation of lipofuscin, or "age pigment".
For this study, we aimed to investigate the expression of autophagic markers and the accumulation of pathologic proteins such as APP and lipofuscin in aged bovine brains. Microscopic findings in the brains of our aged bovines are similar to those previously described in old animals of other species as well as in cattle. In this study, the age-dependent intraneuronal accumulation of lipofuscin is one of the most striking features of aged brains. This finding is not actually new, as it has been described for more than 150 years. In the past, lipofuscin was generally thought to be an innocent end product of oxidation which has no significant influence on cellular activities, but in the last decade several authors have investigated about the possible detrimental and pathogenic potential of this material.
The so-called "mitochondrial-lysosomal axis theory of aging" tries to explain the possible relationship between lipofuscin accumulation, decreased autophagy, increased Reactive Oxygen Species (ROS) production, and mitochondrial damage in senescent long-lived postmitotic cells. According to this theory, in senescent cells lysosomal enzymes are directed towards the plentiful lipofuscin-rich lysosomes and, subsequently, they are lost for effective autophagic degradation because lipofuscin remains non-degradable. The consequences are a progressive impairment of autophagy and the gradual accumulation of damaged mitochondria, other organelles and misfolded proteins that lead to neurodegeneration. Unfortunately, our results cannot support a direct association between lipofuscin accumulation and autophagy impairment in aged bovine brains. According to recent scientific literature, we can only hypothesize that progressive and severe lipofuscin accumulation may irreversibly lead to functional decline and death of neurons by diminishing lysosomal degradative capacity and by preventing lysosomal enzymes from targeting to functional autophagosomes.
Further studies are indeed necessary to better understand how lipofuscin accumulation can influence the neuronal autophagic and apoptotic pathways in bovine brains. It would be interesting to perform double-staining techniques in order to show whether lipofuscin is directly related to autophagic and apoptosis markers and/or to pathologic protein deposition. Unfortunately, to our knowledge, a specific antibody for lipofuscin is not available since this complex substance is mainly composed of cross-linked protein and lipid residues. Alternatively, combined histochemical and immunohistochemical staining protocols can be performed to simultaneously localize lipofuscin and the antigen of interest. However, since lipofuscin progressively accumulates throughout the life of neurons, this combined immunohistochemical/histochemical protocol is not perfectly indicated to investigate the mechanism and relative timing of intraneuronal lipofuscin accumulation and the deposition of other proteins. Primary cultured neuronal cells exhibit, in vitro, a variety of features that are frequently observed in physiologically aged neurons in vivo, including lipofuscin accumulation. Thus, long-term aging culture of primary cultured neurons would be a remarkable model to unravel, at least in part, the molecular mechanisms behind lipofuscin accumulation and its pathological effects on neuronal cells.