SENS-Diagnostics. Intracellular "debris" biomarkers

Throughout the life of the organism, components are formed in the cells, which for various reasons become unable to properly perform their physiological functions. Structures such as, for example, old defective mitochondria, become intracellular "debris". If such ballast were constantly accumulated inside the cell, this would make it impossible for normal intracellular processes to occur and would lead to cell death. To prevent this from happening, there are special “incineration plants” in the cells - lysosomes.

Lysosomes are single-membrane organelles with a diameter of 0.2 to 2 microns. In order to accommodate the cell structures intended for degradation, lysosomes are able to take on many different forms. On average, there can be several hundred lysosomes in a single cell. The degradation of cellular components and macromolecules intended for utilization occurs in the lysosome under the influence of special splitting enzymes (about 60 different types in total), the main of which is acid phosphatase.

Over time and under the influence of various factors, a lysosome can begin to cope worse with its tasks. What leads to the accumulation of "intracellular debris" that interferes with the life of the cell. This problem is particularly relevant for postmitotic cells such as heart muscle cells and neurons. The progressive accumulation of intracellular "garbage" in leads to disruption of the normal functioning of the cells, which can lead to the emergence of diseases and accelerated aging.


Lipofuscin deposits in the heart cell. Lf - lipofuscin, m - mitochondria, mf - myofibrill

A typical example of such a clogged cell can serve as our immune guard - a macrophage. It is known that macrophages, one of the functions of which is to protect the inner walls of the artery, are simultaneously a key element in the development of atherosclerosis. In a healthy and young body, macrophages quite well cope with their task, absorbing and degrading substances dangerous for the arteries, such as modified lipoproteins. But if such substances in the arteries become larger than the macrophage can digest, toxic materials accumulate inside it. And this leads to dysfunction of the macrophage and its transformation into a foamy cell, which already directly participates negatively in the development of atherosclerosis.

The authors of the SENS concept see the solution to the problem of age and pathological accumulation of intracellular wastes in the modification of lysosomes. They suggest that since the main problem is the inability of the lysosomes to destroy part of the intracellular inclusions, a logical solution is to supply the lysosomes of new enzymes that can cope with this task. As it is known today, such enzymes exist, for example, in soil bacteria and fungi that decompose organic residues. Thus, the idea of ​​SENS is to find the enzymes used by these organisms to digest lysosomal wastes, change them so that they can work in the human lysosome environment, and then deliver them to the cells.

At first glance, this approach may seem technically difficult, but there is already direct evidence of its viability. They were obtained in the treatment of genetic pathologies, the so-called. lysosomal storage diseases (Lysosomal Storage Diseases), such as Gaucher disease, using “enzyme replacement therapy”. These diseases are of a genetic nature, with the result that lysosome enzymes do not work as they should. And today, a number of such diseases are successfully treated by injecting patients with lysosomal enzymes modified to move through the cell membrane. Further work in this direction continues, using gene therapy, so that the cells overloaded with waste begin to produce the enzymes necessary for the degradation of the garbage.

One type of intracellular "garbage", which can also serve as a biomarker of aging, is today considered lipofuscin granules. Lipofuscin is a pigment consisting of oxidized and cross-linked proteins and lipids, which complicates its degradation and elimination from cells. Lipofuscin was discovered by R. Virkhov in 1847. But despite this, there is still no clear understanding of either the mechanisms of its formation, nor its role in the body in general and in the aging process in particular.

The term “lipofuscin” was introduced into scientific circulation in 1912 and literally means “dark fat”, due to the brown color of the pigment. Prior to this, lipofuscin granules were called “ceroids,” according to the place of their formation, in hepatocytes, in the liver prone to cirrhosis. The size of the granules varies on average from 0.5 to 1.5 microns. Today, lipofuscin is found in the cells of all living species, from protozoans to primates. What casts doubt on the purely pathological role of lipofuscin, somehow fixed by evolution in all living organisms. On the other hand, lipofuscin may take part in the programmed death of organisms, which describes the theory of phenoptosis proposed by academician V. Skulachev.

The composition of lipofyscin includes fats (20-50%), proteins (30-60%). Fats are mostly represented by phospholipids (kefalin, lecithin, sphingomyelin), and also cholesterol, triglycerides, and products of peroxidation and polymerization of fatty acids. The composition of lipofuscin granules can include all known amino acids, the proportion of which depends on the organ from which lipofyscin was isolated. But in the greatest quantity in all lipofuscin granules four amino acids are represented: glycine, valine, alanine and proline.

Some lipofuscin proteins belong to protein-enzymes, among which the largest amount of acid phosphatases (the main enzyme of lysosomes) and non-specific esterases. In addition, nitrogenous bases, benzene derivatives, phenolic compounds, and inorganic components — carbon, nitrogen, phosphorus, sulfur, magnesium, and aluminum — have been found in lipofyscin granules [1].

How is lipofuscin formed in the cells? Today there is no exact answer to this question. The presence of lysosomal enzymes (acid phosphatase) in the composition of lipofuscin suggested that lipofuscin may appear due to oxidative stress. As a result of which, “resident bodies” accumulate in the cell - products of lipid oxidation and peroxidation that are not amenable to degradation by lysosomal enzymes. Mitochondrial enzymes, fragments of mitochondria and the endoplasmic reticula were also detected in lipofuscin granules. Therefore, the nature of the accumulation of lipofuscin in cells may also be associated with the destruction of cellular organelles that have not been utilized by lysosomes [2]. Thus, mitochondria are most susceptible to degradation into lipofuscin granules (mitolipofuscin) [3].

It is described that iron enhances the formation of lipofuscin, presumably by increasing the peroxidation of lipids caused by iron. Some researchers secrete a separate form of lipofuscin, especially rich in iron - hemosiderin. With aging, the dysfunction of mitochondria and the associated oxidative stress has a strong effect on lipofuscin accumulation (and here you can see the intersection of two SENS sections - mytoSENS and lysoSENS). Thus, excessive production of defective mitochondria of hydrogen peroxide causes peroxide to diffuse into lysosomes overloaded with iron, and increases the formation of lipofuscin. Mitochondria also contain ferritin, which can be degraded by autophagy, which provides an additional source of free iron to further enhance the production of reactive oxygen species. Including the reduction of iron ions Fe2 + to Fe3 + in the Fenton reaction. Increased oxidative stress catalyzed by iron causes peroxidative damage to lysosomes (permeability of their membranes), which leads to iron leakage, and, in turn, increases the total pool of iron. And here we see a cascade of cross-catalytic events characteristic of pathologies: mitochondrial dysfunction and oxidative stress leads to a chain of reactions, the result of which is an increase in lipofuscin accumulation. And this cascade of events is directly associated with a special form of cell death - ferroptosis, which occurs against the background of a deficiency of one of the antioxidants, glutathione, and under conditions of excess iron and oxidative stress [4]. causes peroxidative damage to lysosomes (the permeability of their membrane), which leads to iron leakage, and, in turn, increases the total pool of iron. And here we see a cascade of cross-catalytic events characteristic of pathologies: mitochondrial dysfunction and oxidative stress leads to a chain of reactions, the result of which is an increase in lipofuscin accumulation. And this cascade of events is directly associated with a special form of cell death - ferroptosis, which occurs against the background of a deficiency of one of the antioxidants, glutathione, and under conditions of excess iron and oxidative stress [4]. causes peroxidative damage to lysosomes (the permeability of their membrane), which leads to iron leakage, and, in turn, increases the total pool of iron. And here we see a cascade of cross-catalytic events characteristic of pathologies: mitochondrial dysfunction and oxidative stress leads to a chain of reactions, the result of which is an increase in lipofuscin accumulation. And this cascade of events is directly associated with a special form of cell death - ferroptosis, which occurs against the background of a deficiency of one of the antioxidants, glutathione, and under conditions of excess iron and oxidative stress [4]. mitochondrial dysfunction and oxidative stress leads to a chain of reactions, the result of which is an increase in lipofuscin accumulation. And this cascade of events is directly associated with a special form of cell death - ferroptosis, which occurs against the background of a deficiency of one of the antioxidants, glutathione, and under conditions of excess iron and oxidative stress [4]. mitochondrial dysfunction and oxidative stress leads to a chain of reactions, the result of which is an increase in lipofuscin accumulation. And this cascade of events is directly associated with a special form of cell death - ferroptosis, which occurs against the background of a deficiency of one of the antioxidants, glutathione, and under conditions of excess iron and oxidative stress [4].

At various times, suggestions have been made about the possible physiological role of lipofuscin. Thus, in the composition of the lipofuscin granules, except for proteins and lipids, Soviet researchers V.N Karnaykhov and AB. The substances that are actively involved in cell metabolism, carotenoids, were found in Tatariunas [5, 6]. In this regard, it was suggested that lipofuscin performs some functions. Presumably, he can participate in the production of energy under conditions of hypoxia in energy-intensive fabrics, for example, in muscle tissue. For example, it is known that in muscle cells the amount of lipofuscin increases under the influence of heavy physical exertion.

But most researchers are of the opinion on the age-dependent accumulation of lipofuscin in the cells, which makes it possible to use it as an aging biomarker. Perhaps the most clearly formulates the relationship of lipofuscin with aging is the mitochondrial-lysosomal theory of aging. According to which the age-related accumulation of lipofuscin, which is blocking lysosomes, is closely associated with a progressive violation of autophagy, oxidative stress and dysfunction of mitochondria. What causes gradual age-related accumulation of damaged mitochondria, other organelles and damaged proteins, and, as a consequence, cell dysfunction of postmitotic cells, organ dysfunction and aging [7].

It is known that with age there is an increase in the accumulation of lipofuscin in different cells: in the cells of the brain, heart, retina, skeletal muscles, skin [8]. For non-dividing cells, age congestion with lipofuscin should be a big problem. Thus, the negative role of this pigment in neurodegenerative processes and its possible relationship with neuropathologies (Alzheimer's, Parkinson's, and others) have been described [9]. Therefore, the use of lipofuscin as an aging biomarker seems quite logical.

To identify it in cells today, several histochemical reactions are used: the Hueku reaction (staining with Nile blue dye), the Schmorl reaction (lipofuscin and another pigment melanin are stained in dark blue), the Zill-Nilson method (staining in bright red Carbol-Fuchsin) [10].

One of the popular methods for determining lipofuscin today is staining with Sudan black-B dye (Sudan-Black-B, SBB) and its biotin-related analog (called GL 13), which have shown their high efficiency in detecting lipofuscin [11, 12].

In 2015, Chinese researchers described an effective method for measuring lipofuscin in saliva and blood plasma. Comparative analysis of lipofuscin levels in saliva and in blood in young and old people showed a significant correlation in the direction of its increase with age [13]. That allows you to use this method to determine lipofuscin as a biomarker of aging of the body.

Unfortunately, at the moment there are no effective methods for either slowing down the accumulation of lipofuscin or removing lipofuscin granules. So, patented by Ulrich Schrärmeyer (Ulrich Schraermeyer) in 2008, the method of treatment of Stargardt's disease (associated with the accumulation of lipofuscin in the retinal pigment epithelium) with the help of tetrahydropyridoethers (tetrahydropyridoethers), although it showed encouraging results in the monkey monkeys (tetrahydropyridoethers), it showed encouraging results in the monkey monkeys (tetrahydropyridoethers), which showed encouraging results in the monkey's tetrahydropyridoethers. use in clinical practice [14].

There is no news about the effects of hyperbaric oxygenation on the content of lipofuscin in the brain [15]. The effectiveness of melatonin, isotretinoin, beta-cyclodextrin - the “solvent” of both lipofuscin and atherosclerotic plaques, and other drugs requires further research [16-18].

Review authors: Denis Odinokov, Alexey Rzheshevsky.

To be continued ...

In the next, 3 parts, we will talk about the biomarkers of protein aggregates associated with age-related neuropathology.