Age-related diseases and mitochondria

Diseases and changes in cellular metabolism

Alexey Rzheshevsky, "Biomolecule"

Neurodegenerative and oncological diseases are the most common age–related pathologies after heart and vascular diseases. Studies show that these pathologies are closely related to energy metabolism and mitochondrial dysfunction. A detailed and large-scale study of changes in cellular metabolism during the development of these pathologies contributes to the development of more advanced diagnostic tools that allow detecting the disease at its earliest stage.

Probably, everyone who begins to get acquainted with the amazing organization of our cells has a feeling of admiration for the incredible complexity of the intracellular world. Every second, complex and strictly coordinated processes take place in billions of our cells. And one of these very important processes is the production of the main energy molecule in the mitochondria – adenosine triphosphate, or ATP. Today it is already well known that the work of mitochondria is very closely related to health and life expectancy [1]. Mitochondria produce energy to support life, but at the same time they also serve as the main sources of reactive oxygen species, an excess of which is detrimental to cells.

Energy exchange

Any living organism is in constant communication with the environment, continuously exchanging matter with it. There are three stages in this process:

• intake of substances;
• metabolism;
• allocation of end products.

Intracellular metabolism, in turn, includes two types of reactions: catabolism and anabolism.

Catabolism is the process of splitting and oxidation of organic molecules, leading to the formation of heat and energy molecules, ATP. It is due to the constant production–splitting of the latter that the calories we eat are sent "to the address": hydrolysis of two high-energy (macroergic) bonds in ATP molecules provides energy for all kinds of synthetic and transport processes in cells. At the first stage of catabolism, under the influence of digestive enzymes, complex organic compounds (proteins, polysaccharides, fats) break down into simpler ones – amino acids, monosaccharides, fatty acids and glycerin – which the cell uses for anabolism reactions (plastic metabolism) and energy production. Amino acids are used for protein synthesis. Fatty acids perform an energy function, are part of cell membranes and serve as a substrate for the synthesis of eicosanoids.

At the second stage, glycolysis occurs – the cleavage of glucose molecules (Fig. 1) to pyruvic acid (PVC). The further course of reactions depends on the presence or absence of oxygen in the cell. If there is no oxygen (anaerobic process), then PVC in microorganisms and plants will turn into ethanol, and in animals – into lactic acid [2]. Anyone who subjected themselves to heavy physical exertion could feel the end result of anaerobic metabolism in the form of pain and stiffness in the muscles due to lactic acid accumulated in them.

Drawing 1. Glycolysis reactions. At 10 stages of glycolysis (five preparatory and five stages of ATP synthesis), two three-carbon molecules of pyruvic acid are formed from a hexacarbon glucose molecule. The energy obtained from the splitting of glucose is stored in the "energy currency" of the cell – two molecules of ATP and two molecules of NADP (Wikipedia).

If there is oxygen in the cell, PVC will split into carbon dioxide and water and also release the energy contained in the carbohydrate molecule. This process is called aerobic cellular respiration and takes place in special organelles – mitochondria. Oxidation in mitochondria provides much more energy than glycolysis.

Mitochondria and ATP production

Mitochondria are a real biological miracle created by evolution. Despite their very small size (there can be more than 1000 mitochondria in one cell), these organelles are extremely complex in their organization (Fig. 2). They are elongated "bubbles" surrounded by two membranes. It is believed that mitochondria were formed as a result of absorption by archaea-phagocytes of purple photosynthetic bacteria, which, adapting to an excess of oxygen, mastered aerobic respiration [3], [4]. Mitochondrial membranes consist of lipids and hydrophobic, water-insoluble proteins. (Here we describe the structure of mitochondria in such detail not by chance, but in order to make it clear later how their normal work and dysfunction affect health.)

Figure 2.

The structure of the membranes is very important for the breathing process. The outer membrane of the mitochondria is smooth, and the inner membrane is repeatedly folded. These folds (or crystals) allow you to increase the working area of the membrane, which is necessary to accommodate the entire complex of proteins that carry out respiration there. First, carbon atoms of carbohydrates, fatty acids and amino acids are oxidized to CO ₂ (glycolysis, Krebs cycle and β-oxidation of fatty acids), and the electrons obtained in this way are used to form NADP. Next, NADP is oxidized by molecular oxygen to form water. The NADP-oxidase reaction is accompanied by the release of a very large amount of free energy (about 1.1 eV when transferring one electron from NADP to oxygen), which can be stored by the respiratory chain in the form of a transmembrane difference in the electrochemical potentials of H+ ions (protons).

The work of respiratory proteins-enzymes is similar to the work of pumps: by transferring electrons to each other, they pump protons into the intermembrane space (see video 1). As a result, the inner membrane of the mitochondria is charged like a capacitor. Potentials are created: electric (positive charges are outside the mitochondrial membrane, negative charges are inside the organelle) and chemical (there is a difference in proton concentrations: there are fewer of them inside the mitochondria, more outside). It is known that the electric potential on the mitochondrial membrane, which serves as a good dielectric, reaches 200 mV with a membrane thickness of only 10 nm [5]. For comparison, the action potential on the membranes of nerve cells during signal transmission reaches only 30 mV.

Video 1. How Mitochondria works

Having accumulated in the intermembrane space, protons, like an electric current, rush back into the mitochondria – to where their concentration is lower. However, they can only pass through special channels of ATP synthase embedded in the inner membrane: the proton channel (rotor) of this enzyme is fixed in the membrane, and the catalytic complex sticks out into the mitochondria, into the matrix (Fig. 3). The flow of protons spins the rotor like a river spinning a watermill. As a result, the rotor rotates at an incredible speed – 300 revolutions per second (see video 2). And it is this rotation that leads to the formation of a high–energy molecule - ATP [6]. It is estimated that about 40 kg of ATP is synthesized and consumed per day in the adult body, while the life of each molecule is very short.

Figure 3. The scheme of the mitochondrial respiratory chain (The presence of supercomplexes
in the respiratory chain, electron transfer is provided by the SCAFI protein, elementy.ru ).

Video 2. The work of ATP synthase in the mitochondrial membrane

All of the above is directly related to aging. The fact is that in the process of respiration, enzymes do not work quite "cleanly", and as a result, by–products are formed - reactive oxygen species (ROS). While a person is young and healthy, the ROS formed in the mitochondria do not pose a tangible threat to him, since they are easily neutralized by the body. But when a person gets old, leads an unhealthy lifestyle or has a genetic predisposition to certain diseases, his defense systems fail, collapsing one after another.

Fatty acids and mitochondrial dysfunction

The fact that aging and age-related pathologies are accompanied by mitochondrial dysfunction, which begin to produce less ATP and renew worse, no one doubts. It also turned out that mitochondrial dysfunction and aging are closely associated with an increase in the level of free fatty acids in the blood [7], which is strongly promoted by sedentary and irrational nutrition. Fatty acids, entering the cell, are able to directly reduce the synthesis of ATP, separating oxidation and phosphorylation. This phenomenon related to the thermoregulation of the body was discovered six decades ago by Academician Skulachev and his colleagues [8]. A decrease in ATP synthesis, in turn, triggers several negative chain reactions associated with age-related diseases and aging in general.

And that's what happens. An increase in the level of free fatty acids in the body leads to insulin resistance: insulin-dependent cells will stop responding to this hormone. As a result, the absorption of glucose and fatty acids is disrupted, the oxidation of the latter worsens. The fact is that the high level of insulin characteristic of the state of insulin resistance activates a cascade of reactions that blocks the work of the enzyme carnitine palmitoyltransferase I (SRT1) involved in the transfer of fatty acids into the mitochondria [9]. Because of this, ATP synthesis worsens, and fatty acids accumulate in the cytoplasm of cells, causing the effect of lipotoxicity. In addition to insulin resistance, excess fatty acids in the body causes resistance to another "food" hormone – leptin. And because of this, the function of one of the main participants in the biogenesis (renewal) of mitochondria suffers - the coactivator of the gamma receptor activated by peroxisome proliferators (PGC-1α). As a result, mitochondria produce less ATP, age, die and provoke cell death by apoptosis [10].

And finally, excess fatty acids cause stress of the endoplasmic reticulum (EPR) – an intracellular organoid involved in protein synthesis and many other processes. Under EPR stress, calcium ions are released into the cytoplasm, which can cause mitochondrial dysfunction and death [11]. Calcium ions can accumulate in the cell for another reason – due to the deterioration of the ion pumps pumping calcium out of the cell. And the reason for this is the disruption of mitochondria, accompanied by a decrease in ATP synthesis, without which ion pumps refuse to work. As a result, a vicious circle is formed: a decrease in ATP production leads to mitochondrial dysfunction, which further reduces ATP production, etc.

Fatty acids, ceramides and neuronal damage

As it turned out, excess fatty acids and mitochondrial dysfunction are directly related to the occurrence of age-related neurodegenerative pathologies. I must say that the cells of the nervous system are the most vulnerable to age–related oxidative stress and a decrease in ATP synthesis. Such exceptional sensitivity of neurons to energy deficiency and increased generation of ROS is explained by several reasons.

Firstly, the nervous tissue, by virtue of its physiology, needs the greatest oxygen consumption. As a result, intensive oxidative metabolism occurs in the mitochondria of neurons, which becomes the main cause of increased generation of ROS.

Secondly, due to the fact that the membranes of neurons contain a lot of unsaturated fatty acids, they are easily subjected to lipid peroxidation. Since the activity of antioxidant systems in brain tissue is lower than in other organs, and the number of some antioxidant enzymes decreases with age, it becomes clear why the cells of the nervous system are most sensitive to oxidative damage [12].

Currently, several factors that damage neurons are known. Among them are proteins forming intracellular aggregates (β-amyloid protein and others), as well as ceramides and lipofuscin. Their number is primarily affected by the excess of fatty acids in the body. An aggravating circumstance in this case is the excessive content of saturated acids (palmitic and stearic) in the diet. All this together serves as a powerful stimulus for the development of various neurodegenerative diseases, such as Alzheimer's disease [13], [14].

But how can palmitic acid contribute to neurodegeneration? It was found that due to the excess of this acid, ceramides accumulate, which are involved in the regulation of terminal differentiation, proliferation and apoptosis of neurons. Through several chemical reactions, they affect the regulators of the cell cycle, increasing the concentration of kinase inhibitors p21/SDI1 and p27/KIP1. Thus, ceramides stop the cell cycle, which, in turn, activates the main "guardian of the genome" – the p53 protein – and "sends" apoptosis to the cell [15]. In addition, during the degradation of ceramide, a substance sphingosine is formed, which has a cytotoxic effect and can cause both apoptosis and cell necrosis. But that's not all. It was found that the accumulation of saturated fatty acids (palmitic and stearic) stimulates special brain cells (astroglia) for endogenous (internal) synthesis of ceramides. These produced ceramides trigger a chain reaction of the following type: ceramides → increased secretion of proinflammatory cytokines and nitric oxide → increased production of ROS and oxidative stress → activation of stress-regulated kinases (CDK5 and GSK-3) in neurons → formation of β-amyloid protein and hyperphosphorylation of τ-protein [16].

Neurodegenerative pathologies and mitochondrial dysfunction

Today, the most important and most common neurodegenerative pathologies are Alzheimer's, Parkinson's, Huntington's diseases, as well as amyotrophic lateral sclerosis. Their occurrence is associated with structural changes in various proteins, leading to the formation of intracellular aggregates. Such proteins include:

• beta-amyloid precursor, presenilins 1 and 2 (Alzheimer's disease);
• τ-protein, α-synuclein, parkin (Parkinson's disease);
• huntingtin (Huntington's disease);
• superoxide dismutase-1 (amyotrophic lateral sclerosis);
• ubiquitin , etc .

Alzheimer's disease (AD) is a severe neurodegenerative disease characterized by synaptic dysfunction and neuronal death, which is accompanied by a decrease in cognitive abilities: deterioration of memory and thinking, gradual loss of social and motor skills [17]. The risk zone for the development of the disease is mainly elderly people. Only 1-2% of people under the age of 65 suffer from AD. According to one of the hypotheses of the development of BA – amyloid, – the disease occurs due to the accumulation of beta-amyloid aggregates in the brain. This peptide consists of 39-43 amino acid residues and is a fragment of a large transmembrane protein called beta-amyloid precursor protein (APP). Being in excess, the β-amyloid molecules begin to "stick together" and form insoluble plaques (Fig. 4). It is in this state that the protein disrupts the work of nerve cells and causes symptoms of AD. In AD sufferers, a large number of amyloid plaques and neurofibrillary tangles are found in the affected areas of the brain [18].

Drawing 4. Formation of amyloid plaque in genetically engineered mice (shown by a long arrow). On the 6th day, neuron dystrophy is already visible (short arrow). Amyloid deposits are indicated in blue, neurons are indicated in green. The length of the scale ruler is 20 microns; the images were taken using a multiphoton microscope [33].

However, the amyloid hypothesis is not the only one explaining the occurrence of AD. In 1993, Allen Roses, a professor at Duke University, proposed another hypothesis for the occurrence of AD – a genetic one associated with the APOE gene encoding apolipoprotein E (ApoE). It turned out that inheritance of one of the variants of the APOE gene – APOE4 – increases the chances of getting AD several times. More and more researchers are inclined to think that beta-amyloid is unnecessarily "demonized" and is not the root cause of the development of AD. The failed therapy aimed at clearing the cells of β-amyloid confirms that not everything is completely clear with this disease [19].

Parkinson's Disease (PD) – another severe and quite common age-related neurodegenerative disease. In patients with PD, α-synuclein accumulates in the neurons of the substantia nigra, which forms special granules – Levi's corpuscles. I must say that there is a so-called dementia with Lewy bodies, which is characterized by the accumulation of numerous Lewy bodies in cortical and subcortical neurons and the development of progressive cognitive disorder in the first year of the disease. But it is not yet clear whether to consider this dementia a form of PD or it is more correct to consider it as a separate disease. In the case BP accumulations of Levi's bodies lead to dysfunction of neurons and their death, while damage to brain regions from the so-called nigrostriar dopamine pathway is characteristic. This pathway regulates motor activity, reducing muscle tension. That's why, when dopamine neurons die, patients have corresponding symptoms: an increasing increase in muscle tone and trembling of the hands. In addition to impaired motor functions, PD is characterized by other symptoms associated with sleep disorders, depression, anxiety, visual impairment and slow thinking [20].

Huntington's disease (BH) is also not too rare neurodegenerative disease [21]. As in the case of Alzheimer's disease, the pathogenesis of BH is characterized by the formation of toxic protein aggregates involving mutant forms of proteins that are synthesized in the nervous tissue. But if scientists have questions about the main "culprit" of BA, beta-amyloid, in the case of BH there is much less doubt. It has been established that it is genetic features – polymorphisms of certain DNA sites – that lead to the appearance of pathological forms of the huntingtin protein. Such huntingtin is capable of association with other proteins of the nervous tissue, resulting in the formation of insoluble toxic aggregates that damage the cortex and striatum of the brain. Bursts of involuntary motor activity, emotional disorders and memory loss are typical for BH. At the same time, the normal physiological function of the huntingtin protein in the body remains questionable. It is assumed that it plays some role in embryogenesis [22].

All three mentioned pathologies are most closely related to mitochondrial dysfunction. First of all, it should be noted that its development under the influence of defective proteins specific to neuropathologies has been established in several ways: in vitro (on cell lines and extracellular systems) and in vivo (on transgenic animals). A feedback was also found: it turned out that mitochondrial dysfunction can stimulate the appearance of defective proteins. Thus, a violation of the activity of respiratory complex I leads to the accumulation of hyperphosphorylated τ-protein and α-synuclein in nerve cells [23].

The already mentioned stress of the endoplasmic reticulum was also associated with the accumulation of defective proteins. One of these proteins, α-synuclein, can reduce the activity of proteasomes, which results in EPR stress, increased ROS production and initiation of apoptotic processes. This is because an apoptotic factor, cytochrome C, is released from the mitochondria, which activates the "cell killers" – caspase-9 and caspase-3 [24]. It is believed that at the initial stages of neurodegeneration in AD, the accumulation of β-amyloid and hyperphosphorylation of the t-protein may be physiological mechanisms for protecting the cell from oxidative stress caused by progressive mitochondrial dysfunction. However, with excessive accumulation of these proteins in the cell, the mitochondria malfunction. Thus, in patients with AD, it was found that β-amyloid accumulates in mitochondria and disrupts the reactions of glycolysis and the Krebs cycle, activates the production of ROS. Moreover, β-amyloid is able to directly suppress the synthesis of ATP. This is possible due to the structural similarity of the protein with a natural inhibitor of the F(1) subunit of mitochondrial ATP synthase. Beta-amyloid can also interact with the mitochondrial membrane, forming stable complexes with two translocases, TOM40 and TIM23. Such complexes inhibit the import into mitochondria of proteins encoded by the nuclear genome – cytochrome oxidase subunits IV and Vb. To which the organelle responds by increasing the production of aggressive hydrogen peroxide.

But that's not all: the beta–amyloid precursor protein can form pores in the membranes of mitochondria and other organelles, which disrupts the ion balance in the cell and triggers its apoptosis [25]. Also, this protein increases the activity of phospholipase D, as a result changing the phospholipid composition of mitochondrial membranes, increasing the concentration of phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid and disrupting the membranes. It is known that β-amyloid can bind heme, and this leads to heme deficiency in the cell, which disrupts the work of the heme-containing IV complex of the mitochondrial electron transport chain [26].

But not only beta-amyloid is able to negatively affect mitochondria. In experiments with transgenic rodents expressing the human huntingtin gene, aggregation of this protein in mitochondria was detected with the subsequent development of their dysfunction. Another "harmful" protein, α-synuclein, accumulating in the inner mitochondrial membrane, is able to reduce the activity of the respiratory complex I. As a consequence, mitochondria increase the production of ROS [27]. It has also been found that α-synuclein, interacting with mitochondria, can stimulate the release of cytochrome from them C, which means to initiate apoptosis.

In general, we can say that the launch of apoptosis is a characteristic effect of proteins that cause neurodegeneration. They can directly or indirectly affect regulatory proteins associated with apoptosis: p53, Akt, Bad, Bax, Bcl-x(L) and calcineurin [28].

It is also described that the supersynthesis of the beta–amyloid precursor protein leads to damage to the mitochondrial fusion–division system. Mutations of the Parkin gene (PARK2) found in PD patients negatively affect the same system and the utilization of defective mitochondria by autophagosomes. Defective forms of t-protein and huntingtin also interfere with the normal functioning of mitochondria, thereby impairing the energy supply of nerve cell processes and synaptic transmission, causing degeneration of synapses [29].

Thus, proteins involved in the development of neurodegenerative pathologies can contribute to mitochondrial dysfunction through a number of mechanisms. In turn, the dysfunction that has already arisen can aggravate pathological processes, stimulating the appearance of defective proteins and thereby closing the vicious circle of the disease.

The Warburg Effect

And finally, it is worth touching on another point related to pathologies and changes in cellular metabolism. In 1926, German biochemist Otto Warburg compared the rates of lactic acid (lactate) formation in normal and tumor cells. It turned out that tumor cells consume a lot of glucose, while forming lactate. And they do it much faster than normal cells: the malignant tissue in the experiment produced lactic acid eight times more actively than it does in a muscle performing physical work. Warburg found that cancer cells use glycolysis to produce energy regardless of the availability of oxygen (Fig. 5) [30]. In honor of the discoverer, this phenomenon was called the Warburg effect [2].

Drawing 5. Energy supply of normal and cancer cells (metabiolab.com ).
The blue square indicates the glucose entering the cell.

Having discovered this effect, Warburg logically assumed that it could be explained by mitochondrial dysfunction in tumor cells and a violation of oxidative phosphorylation. Today, this point of view is being questioned, since a large number of normally functioning mitochondria are also found in the degenerated tissue. About half of the total energy of tumor cells is obtained from ATP molecules produced in mitochondria [31]. The Warburg effect manifests itself in cells already at the very beginning of their transformation into tumor cells. And this makes it possible to carry out early diagnosis of neoplastic processes: as soon as the cell began to consume glucose on an increased scale, it's time to sound the alarm. These processes can be detected using positron emission tomography using a fluorinated glucose analog, 2-(18F)-2-deoxy-D-glucose.

But why do cancer cells switch to anaerobic glycolysis? Now it is believed that this way they get an advantage, preparing in advance for "hard times" – the development of hypoxia. In addition, this method of energy supply gives cells the opportunity to use glycolysis intermediates for anabolic reactions, enhance their antioxidant defense and repel the immune attack of the body [32].

Thus, changes in glucose metabolism and the appearance of defective proteins and intracellular aggregates may indicate the beginning of the development of pathology. Timely detection of such intracellular processes can play a crucial role in the prevention and treatment of the most common neurodegenerative and oncological diseases. And in order for this to be possible, it is necessary to study the fundamental aspects of pathologies related to the work of mitochondria and energy metabolism. Today, systems have already been developed that allow you to look "deep into" these diseases and even diagnose them at the earliest stage of their development.

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