Life extension: Resveratrol, spermidine and autophagy (1)

Autophagy mediates the increase in life expectancy caused by spermidine and resveratrolArticle by Eugenia Morselli et al.
Autophagy mediates pharmacological lifespan extension by spermidine and resveratrol
published in Aging magazine, December 2009, volume 1, No. 12.
Translated by Evgenia Ryabtseva

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Despite the fact that autophagy is traditionally considered a self-destruction mechanism leading to cell death, evidence is gradually accumulating in favor of the fact that under normal conditions autophagy mediates cytoprotection mechanisms, thereby allowing stressed cells to avoid apopototic or necrotic death. Recent data indicate that autophagy is also involved in pharmacological manipulations that increase life expectancy. The intake of polyamine spermidine from the external environment can increase the lifespan (while inducing autophagy) of yeast, nematodes and fruit flies. Resveratrol can also trigger autophagy in cells of various organisms, increase the lifespan of nematodes and increase the viability of human cells that tolerate metabolic stress. These positive effects are absent under the condition of genetic or pharmacological inactivation of the main autophagy modulators. This indicates that the launch of autophagy is necessary for the realization of cytoprotective and/or aging-slowing effects of spermidine and resveratrol. According to the results of genetic and functional studies, to protect cells and increase life expectancy, spermidine inhibits histone acetylases, while resveratrol activates histone deacetylase sirtuin 1. Although it is not yet clear whether the same histones (or possibly other cellular or cytoplasmic proteins) are indirect targets of spermidine and resveratrol, these data indicate an important role belonging to a decrease in the level of acetylation in the regulation of autophagy activity and a change in life expectancy.

IntroductionAutophagy (from the Greek "self-eating") consists in isolating and destroying with the help of lysosomal enzymes old, superfluous, damaged or located in inappropriate places of the cell organelles and/or cytoplasm fragments [1].

At least three forms of autophagy (macroautophagy, microautophagy and autophagy mediated by chaperone proteins) have been described in the literature, differing in the method of delivery of biological material into lysosomes [2, 3]. This article is devoted to macroautophagy (hereinafter "autophagy"), which is the most important catabolic process used by cells for processing long-lived proteins and organelles, as well as one of the most outstanding cytoprotective mechanisms in the biology of eukaryotic cells [4].

In the process of autophagy, the material intended for degradation is delivered to lysosomes by isolating inside bubbles surrounded by a two-layer membrane, called autophagosomes. The formation of an autophagosome begins with the formation and elongation of the so–called phagophore - a fragment of the insulating membrane separating from the endoplasmic reticulum. The edges of the phagophore close together to form the autophagosome proper, which subsequently merges with the lysosome to form an auto (phago)lysosomes. Ultimately, the contents of the lumen, as well as the inner membrane of the auto (phago)lysosomes (collectively called the "autophagic body") are cleaved by lysosomal hydrolases. The end products of such catabolic reactions are released into the cytoplasm, where they can re-enter the reactions of anabolic and/or bioenergetic metabolism [2, 3, 5, 6].

For the first time at the molecular level, the biochemical cascade underlying autophagy was described for yeast (Saccharomyces cerevisiae) [7, 8]. The results of hundreds of studies on various model organisms, including mammals, have confirmed that the main mechanisms of autophagic isolation and degradation have been preserved during evolution and are mediated by orthologs of a complex of yeast genes called autophagy-related (ATG) genes [7, 8]. Low basic activity of autophagy, providing homeostatic turnover of long-lived proteins and organelles, it is registered in all cells [4]. Moreover, in some cases, the activity of autophagy increases to indicators significantly exceeding the baseline level: 1) when the cell needs to mobilize intracellular nutrients, for example, in conditions of insufficient intake of glucose and/or amino acids, hypoxia or deficiency of growth factors [2, 3]; and 2) when removing potentially dangerous cytoplasmic materials from the cell, including damaged organelles, aggregates of improperly formed protein molecules or introduced microorganisms [9, 10].

Complex regulation of autophagy as a reaction to stressOne of the key regulators of autophagy in human and rodent cells is the mammalian rapamycin target protein kinase (mTOR, its yeast analogue is called TOR), which suppresses autophagy in conditions of sufficient intake of nutrients and growth factors.

Signal transducers, including phosphotidylinositol-3-kinase (PI3K) and Akt, provide a relationship between tyrosine kinase receptors and mTOR activation, thereby suppressing autophagy in response to the intake of insulin, insulin-like growth factor (IGF) and other growth factors [11]. Activation of mTOR as part of complex 1 (mTORC1) – and subsequent suppression of autophagy – can also be mediated by mitogen-activated protein kinases (MAPK); including kinases regulated by extracellular signals (ERK) [12]; Ras-dependent activation of p90 ribosomal S6 kinase [13]; as well as Wnt-mediated signaling mechanism [14]. Other important regulators of autophagy include (the list is not exhaustive): AMP-activated protein kinase (AMPK), which inhibits mTOR with a decrease in ATP levels [15]; eukaryotic translation initiation factor 2-alpha (eIF2a), which reacts to insufficient nutrient intake as well as double-stranded RNA (dsRNA) [16], ERN1 (whose yeast orthologue is known as IRE1) is a protein associated with the endoplasmic reticulum (ER) that simultaneously has kinase and endoribonuclease activity and plays an important role in changing gene expression under stress effects on ER [10, 17]; as well as c-Jun-N-terminal kinase involved in many signaling cascades, activated under stress [18].

The studies conducted by the authors of the article have increased this list by several more points: members of the Bcl-2 protein family containing a single Bcl-2 homology domain, or the so-called BH3-only proteins, which displace (and thus return activity to it) the important autophagy modulator Beclin 1 from inhibitory complexes with Bcl-2 or Bcl-XL [19, 20]; sirtuin 1, reacting to high levels of NAD+, actually acting as a sensor of nutrient availability [21]; tumor suppressor protein p53, whose presence in the cytoplasm inhibits autophagy [22]; I-kappa-bi-kinase complex (IkB kinase or IKK), also necessary for the activation of NF-kB under stress [23, 24]; as well as a receptor for inositol-1,4,5-triphosphate (IP3R) at the ER level [20, 25]. Finally, the positive regulation of autophagy is provided by the activity of transcription factors manifested by E2F1 [26], FOXO3a [27, 28], NF-kB [29], p53 [30, 31] and other molecules. ULK1 and ULK2 (mammalian Atg1 orthologs), as well as Beclin 1 (human Atg6 orthologs) are involved in peak events of the phylogenetically ancient molecular mechanism of autophagy. Beclin 1 functions as an allosteric activator of hVps34 – phosphatidylinositol-3-kinase (PI3K) class III (stimulating the formation/elongation of the phagophore with its product phosphatidylinositol-3-phosphate), and is also a component of a highly dynamic multi-protein complex, which may include various stimulants (for example, UVRAG, Bif-1/endophilin B1, Ambra 1) and/or inhibitors (e.g. Bcl-2, RUBICON) of autophagy [32-36].

In general, autophagy is interconnected with numerous mechanisms that are triggered in response to stressful influences. In some cases, certain proteins and organelles are "labeled" for autophagic isolation. This suggests that some characteristics of the contents of autophagosomes determine its fate, which consists in elimination through autophagy. This fact has been recorded for proteotoxins [37-39], dissociated mitochondria (this process was called "mitophagy") [40-42], peroxisomes (for which the term "pexophagy" was introduced) [43]; damaged ER (eliminated by "reticulophagy") [44]; and pathogens introduced into the body (activating "xenophagy") [45]. In many other cases, autophagy is rather indiscriminate in nature and represents a non-specific response of a cell in a state of transition from a baseline to an induced level. It is very tempting to assume that this transition from a basic to an increased level of autophagy may imply the activation of the main "switch" capable of responding to numerous unrelated mechanisms of stress response and damage detection. Complex molecular switches provide a clear separation between different cell states, including the transition from an undifferentiated to a more differentiated state, the change of phases of the cell cycle, or the "decision" to activate the mechanism of apoptosis [46, 47]. As a rule, such switches integrate a variety of signals transmitted through negative feedback loops (supporting homeostasis and keeping the cell in a certain state), and positive feedback loops (characteristic of a rapid transition from one state to another) [48]. There is a large amount of evidence that positive feedback loops are involved in the mechanism of autophagy. For example, the authors found (1) that autophagy induced by rapamycin (inhibiting mTOR) is accompanied by degradation of p53 protein and activation of IKK; (2) at the same time, pharmacological inhibition of p53 by pythitrin-alpha leads to suppression of mTOR activity and activation of IKK; (3) and also that forced transgenic activation of IKK stimulates degradation of p53 simultaneously with inhibition of mTOR [22-24, 49]. All this indicates the existence of a relationship between inhibition of mTOR, activation of IKK and degradation of cytoplasmic p53, provided by a complex of loops of advanced communication with self-reinforcing activity (Fig. 1), although the molecular aspects of these mechanisms are still unclear.

Figure 1. Molecular composition of a hypothetical autophagy switch.
Regardless of the origin of the primary stress signal, the result of independent activation of molecular complexes formed around IkB kinase (IKK), cytoplasmic p53, mTOR and beclin 1 is the launch of a universal reaction. Within these complexes (and possibly others), reversible posttranslational modification of proteins and/or their movement between complexes occurs, which determines the functioning of the integrator/switch that activates the autophagy mechanism. At the same time, it should be borne in mind that the autophagy switch, apparently, includes several positive feedback loops that determine its (in) activation.

Part 2: Autophagy mediates the increase in life expectancy caused by resveratrol.

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