Autophagy Following Heat Stress: The Role of Aging and Protein Nitration

Abstract

Stress can originate from a variety of sources (e.g. physical, chemical, etc.) and cause protein denaturation, DNA damage, and possibly death. In an effort to prevent such deleterious consequences, most organisms possess one or more ways to counteract or even prevent the harmful effect(s) from a given stressor. Such compensation by an organism is known as a stress response; this involves inhibition of housekeeping genes and subsequent activation of genes associated with the stress response.¹ One of the most widely studied groups of stress response genes is a family of molecular chaperones known as heat shock proteins (HSPs). Work from our laboratory agrees with many other studies showing an age-related decline in stress-induced synthesis of HSPs.² A decline in the availability and/or function of HSPs with age can lead to accumulation of damaged proteins, which in turn damages cells. Recently, our laboratory found a significant increase in mitochondrial damage as well as evidence of increased autophagy in rat hepatocytes following heat stress.³ These results, along with findings of increased protein nitration with age, suggest a major role for reactive nitrogen species (RNS) in both the decline in HSP induction and increased hepatocyte pathology observed in old rats following heat stress.

Oxidative/Nitrative Damage and Aging

As an organism ages, the ability to activate stress response genes can become compromised, thereby dramatically reducing the capacity of an organism to tolerate almost any type of stress challenge. Whether a decrease in the heat shock response is a cause or a consequence of aging is still a matter of debate. Most studies agree that failure to properly induce heat shock proteins (HSPs) is a critical property of aged organisms, leading to the accumulation of damaged intracellular proteins which have been frequently used as a biomarker for aging.⁴ Also believed to play a role in the age-related decline in stress tolerance are reactive oxygen species (ROS) and, more recently, reactive nitrogen species (RNS). ROS and RNS are both physiologic messengers and normal by-products of cellular metabolism. In order to protect against possible injury under physiologic conditions, ROS are kept in check by antioxidant enzymes (AEs). As an organism ages, the AE system can become overwhelmed and ROS/RNS begin to accumulate within cells, leading to a buildup of oxidized macromolecules such as proteins, lipids, and nucleic acids that can cause damage to cells.^5,6 Increased protein damage and oxidation with age are thought to result from decreased efficiency of AEs and inefficient clearing of damaged and/or misfolded proteins by molecular chaperones.

The cellular reaction of any organism in response to ROS/RNS is dependent upon many factors, including the type of cell involved, the specific intracellular site of ROS/RNS production, and the amount and/or species of ROS/RNS produced as well as their duration.⁷ Recently, our laboratory used quantitative electron microscopy (EM) to analyze intracellular damage to hepatocytes from young and old rats following heat stress.³ We found that mitochondria, a principal site for ROS/RNS production, were a major target of injury following heat stress, with overall damage greater in old compared to young animals throughout a time course of recovery following heat stress. Development of a significant number of flocculent (“wooly”) densities, thought to be one of the most reliable early manifestations of irreversible cell injury, cell death, and ensuing necrosis⁸, occurred in old rats at recovery time points that directly correlated with high levels of mitochondrial damage. These results suggest that not only is mitochondrial pathology increased in old hepatocytes following heat stress, but at least some of the damage is irreversible.

Several studies have employed immunogold EM techniques for quantification of specific proteins in subcellular compartments.^9,10,11 Levels of 4-HNE-modified protein adducts (4HNE)¹² and 3-nitrotyrosine (3NT)¹³, both proven to be highly specific markers of lipid peroxidation and protein nitration, respectively, were measured.³ The amount of 4HNE in tissues from old rats was not found to be significantly different compared to young cohorts following heat stress; however, there was an approximate two-fold increase in 3NT levels in old compared to young rats in all subcellular compartments measured, implying some sort of RNS imbalance had developed. The presence of 3NT is considered a pathological condition following stress and most often occurs due to augmented RNS production and/or reduced denitration of RNS by specific enzymes.

Heat stress increases ROS/RNS production within hepatocytes, which in turn can cause damage to intracellular components and/or activate specific signaling pathways and transcription factors such as nuclear factor-kappaB (NF-κB), heat shock factor 1 (HSF1), and p53.⁷ Activation of NF-κB can induce the expression of inducible nitric oxide synthase (iNOS), an enzyme that can amplify the rate of NO^• synthesis by a thousand fold under pathological conditions.¹⁴ Nitric oxide is a lipophilic, highly diffusible free radical that when generated locally within mitochondria can react with superoxide (O₂•⁻) leaked from the respiratory chain to form peroxynitrite (ONOO⁻) which then reacts with protein tyrosine residues to form 3NT (Figure 1).Superoxide is a free radical normally scavenged by superoxide dismutase (SOD) under physiological conditions;¹⁵ however, if NO^• were produced in high enough concentrations following induction of iNOS, there could be irreversible damage to mitochondria from ONOO⁻due to the preferential reaction of O₂•⁻ with NO^•. The remarkable stability of ONOO⁻ and its ability to easily diffuse throughout a cell can result in DNA condensation and damage, inhibition of protein synthesis, lipid and protein oxidation, and protein nitration.¹⁶ Elevated levels of 3NT detected in our previous study most likely resulted from increased amounts of NO^• and subsequent formation of ONOO⁻, which could be responsible for the mitochondrial damage observed within hepatocytes from old rats following heat stress.³ When mitochondrial damage becomes severe, increased amounts of ROS/RNS are produced due to the drastic reduction in efficiency of the mitochondrial respiratory chain, thereby also reducing the total ATP content of a cell. Such decreases in ATP can also be caused by the reversible inhibition of catalase (CAT) and/or the oxidation of glutathione (GSH) by nitric oxide (NO^•). Whatever the cause(s) of decreased ATP production, particularly in hepatocytes from old rats following heat stress³, failure to induce HSPs could result.

Figure 1.

Proposed age effects of increased ROS/RNS levels within hepatocytes following heat stress. Various signaling pathways can be activated by ROS/RNS increases. Activation of the transcription factor nuclear factor-kappaB (NF-κB) can cause the induction of inducible nitric oxide synthase (iNOS) expression, which in turn will dramatically increase levels of intracellular nitric oxide (NO^•). Under pathologic conditions, NO^• can outcompete superoxide dismutase for reaction with superoxide (O₂•⁻) and form peroxynitrite (ONOO⁻). ONOO⁻ is remarkably stable and can cause injury to mitochondria by lipid and protein oxidation, DNA damage, and protein nitration.¹⁶ ONOO- reacts with protein tyrosine residues to produce 3-nitrotyrosine (3NT). The presence of 3NT in young animals is reversible and can lead to homeostasis, whereas protein modifications in old animals may cause irreversible protein damage. Induction of autophagy by an unknown transcription factor is a second signaling pathway potentially activated by increased ROS/RNS following heat stress. In young organisms, the autophagic process most often involves efficient recycling of sequestered material, resulting in the cell returning to a homeostatic state. In contrast, aged organisms have a decrease in the efficiency of protein degradation mechanisms, causing accumulation of lipofuscin. ROS/RNS from degraded cellular material (e.g. mitochondria) and iron from both lipofuscin and degraded metallo-proteins allows Fenton chemistry to occur within lysosomes. This in turn produces even more ROS/RNS which can damage the lysosome and inhibit its function, causing an even greater accumulation of undigested lipofuscin and potential leakage of acid hydrolases into the cytosol and production of more ROS/RNS. A third pathway involved following heat stress is activation of the heat shock factor 1 (HSF1) transcription factor. HSF1 binds to the heat shock element (HSE) to induce expression of HSP70 and initiate repair or degradation of damaged/misfolded proteins, a pathway believed to occur optimally in younger organisms. In older organisms, low levels of HSP70 induction and/or function can lead to the accumulation of damaged proteins and formation of aggregates, which in turn produce high levels of ROS/RNS.

Age-related Decline in HSP Induction

Initiation of the heat shock response confers stress tolerance and protection of cellular components, which can ultimately assure the survival of an organism. When an organism is challenged by any of several internal or external stress stimuli, ROS/RNS are produced. When the stressor is heat, an acute state of increased oxidative/nitrative stress leads to the massive induction of HSPs by activation of the transcription factor HSF1 (Figure 1). HSF1 is a long-lived protein monomer found in a wide variety of cell types and is activated by heat stress in an attempt to combat protein aggregation. In fact, lack of HSF1 gene expression was found to cause accelerated damage and an approximate 40% shortening of lifespan in Caenorhabditis elegans.¹⁷ HSF1 binds to a conserved DNA sequence, the heat shock element (HSE), and induces the expression of HSP70, a molecular chaperone that promotes the correct refolding of denatured or unfolded proteins damaged by stress and can also act as a chaperone in the ubiquitin-proteasome degradation pathway.¹⁸ Previous results showed a decrease in hepatic HSP70 protein expression in old rats compared to their young counterparts.² These results agreed with findings from another research group that reported a 50–70% decrease in HSF1 binding activity to DNA (i.e. the HSE) in old compared to young rats following heat stress, which correlated with a subsequent decrease in HSP70 transcription.¹⁹ This group also showed that HSF1 protein levels were two- to three-fold higher in old rats, yet the synthesis of HSF1 in hepatocytes from old rats compared to young was reduced by approximately 50%, indicating the rate of HSF1 degradation is also reduced with advanced age.¹⁹ Cumulatively, these findings suggest the thermostability of HSF1 becomes altered with age, possibly due to a posttranslational modification.¹⁹ Any alteration in HSF1 could seriously compromise the ability of aged organisms to respond to stress and subsequent induction of HSP70 expression. Failure of an aged organism to properly induce the expression of HSPs would ultimately lead to the buildup of oxidatively-modified proteins and formation of denatured protein aggregates; these aggregates in turn increase the level of intracellular stress by production of ROS/RNS.¹⁸ Accumulation of such lipid-protein aggregates (i.e. lipofuscin) was observed in our previous study.³

The Role of Autophagy

Macroautophagy is a catabolic phenomenon of ever-increasing interest responsible for safeguarding a cell from significant injury during periods of stress. The process of macroautophagy involves specific steps of protein degradation which include formation of a limiting membrane (most likely derived from the endoplasmic reticulum in mammals²⁰), isolation of cytosolic components and/or whole organelles within the membrane to form a double membrane vesicle known as an autophagic vacuole (AV) or autophagosome, and ending with fusion of the AV with a lysosome and subsequent degradation of the AV contents. It has been proposed that augmented autophagic removal of damaged proteins and organelles is yet another mechanism employed by cells to protect against the harmful effects of oxidative stress.²¹ This was of particular relevance to our previous study where we found that hepatocytes from old rats demonstrated increased levels of oxidative/nitrative injury following heat stress, along with a significant increase in overall autophagy (i.e., the sum of AVs, autolysosomes, and lipofuscin).³ Brunk and Dalen²² suggested a period of “recuperative” autophagy following oxidative injury evidenced by an increase in the number of AVs in the affected cells. A caveat in this system is that autophagy, like so many other cellular response systems, becomes much less efficient with age.²³ While we observed a significant increase in overall autophagy in both young and old rats 30 min post heat stress compared to contols³, the overwhelming autophagic stage present in hepatocytes from young rats was the AV stage, while old rats at the 30-min recovery point showed more prevalent autolysosomes with only a slight noticeable increase in the number of AVs.

If a physiologic or pathologic stress were mild and the organism were of a young age and sufficient health, an increase in the number of AVs would signify that the organism’s cells were compensating for the effects brought on by the stressor (i.e., increased ROS/RNS induced by heat) and would most likely return to a homeostatic state (Figure 1). In senescent organisms there is an overall decrease in autophagic efficiency, a phenomenon that has been shown to be temperature-sensitive.²⁴ Reduced turnover of damaged proteins leads to decreased induction of AV formation and an increase in the number of autolysosomes present due to inefficient protein degradation. Protein aggregates (i.e., lipofuscin) begin to accumulate in lysosomes where they generate even more ROS/RNS, which through Fenton chemistry (with free iron from degraded metallo-proteins) produces more lipofuscin.²⁵ Such a build-up of lipofuscin within lysosomes would eventually inhibit their function and likely lead to autophagic failure.²¹ This autophagic inefficiency could lead to a build-up of oxidatively modified proteins in the cytoplasm of aged organisms that are now unable to be degraded by the autophagic system. This would place increased demand on other protein degradation systems (i.e. the HSPs); however, with age comes decreased efficiency of these systems as well, leaving cells in senescent organisms with few options in their fight to combat the injurious effects brought on by physiologic and pathologic stress from ROS/RNS.