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Aging results in increased autophagy of mitochondria and protein nitration in rat hepatocytes following heat stress.

ABSTRACT: The natural breakdown of cells, tissues, and organ systems is a significant consequence of aging and is at least partially caused by a decreased ability to tolerate environmental stressors. Based on quantitative ultrastructural analysis using transmission electron microscopy and computer imaging, we show significant differences in hepatocyte morphology between young and old rats during a 48-hr recovery period following a 2-day heat stress protocol. Mitochondrial injury was greater overall in old compared with young rats. Autophagy was observed in both young and old rats, with autophagy greater overall in old compared with young hepatocytes. Lipid peroxidation and protein nitration were evaluated by localization and quantification of 4-hydroxy-2-nonenal (4-HNE)-modified protein adducts and 3-nitrotyrosine (3-NT) levels, respectively. Levels of 3-NT but not 4-HNE-protein adducts were significantly elevated in hepatocytes of old rats in comparison with young at 90 min after heat stress, suggesting a major role for reactive nitrogen species in the pathology observed at this time point. These results show a differential response of hepatocyte mitochondria to heat stress with aging, as well as greater levels of both autophagic and nitrative damage in old vs young hepatocytes. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

SUBMITTER: Oberley TD

PROVIDER: S-EPMC2386765 | biostudies-literature | 2008 Jun

REPOSITORIES: biostudies-literature

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Aging results in increased autophagy of mitochondria and protein nitration in rat hepatocytes following heat stress.

Oberley Terry D TD Swanlund Jamie M JM Zhang Hannah J HJ Kregel Kevin C KC

The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 20080331 6

The natural breakdown of cells, tissues, and organ systems is a significant consequence of aging and is at least partially caused by a decreased ability to tolerate environmental stressors. Based on quantitative ultrastructural analysis using transmission electron microscopy and computer imaging, we show significant differences in hepatocyte morphology between young and old rats during a 48-hr recovery period following a 2-day heat stress protocol. Mitochondrial injury was greater overall in old ...[more]

PMID: 18379016

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Project description:BackgroundHeat stroke is the outcome of excessive heat stress, which results in core temperatures exceeding 40°C accompanied by a series of complications. The brain is particularly vulnerable to damage from heat stress. In our previous studies, both activated microglia and increased neuronal autophagy were found in the cortices of mice with heat stroke. However, whether activated microglia can accelerate neuronal autophagy under heat stress conditions is still unknown. In this study, we aimed to investigate the underlying mechanism that caused neuronal autophagy upregulation in heat stroke from the perspective of exosome-mediated intercellular communication.MethodsIn this study, BV2 and N2a cells were used instead of microglia and neurons, respectively. Exosomes were extracted from BV2 culture supernatants by ultracentrifugation and then characterized via transmission electron microscopy, nanoparticle tracking analysis and Western blotting. N2a cells pretreated with/without miR-155 inhibitor were cocultured with microglial exosomes that were treated with/without heat stress or miR-155 overexpression and subsequently subjected to heat stress treatment. Autophagy in N2a cells was assessed by detecting autophagosomes and autophagy-related proteins through transmission electron microscopy, immunofluorescence, and Western blotting. The expression of miR-155 in BV2 and BV2 exosomes and N2a cells was measured using real-time reverse transcription polymerase chain reaction. Target binding analysis was verified via a dual-luciferase reporter assay.ResultsN2a autophagy moderately increased in response to heat stress and accelerated by BV2 cells through transferring exosomes to neurons. Furthermore, we found that neuronal autophagy was positively correlated with the content of miR-155 in microglial exosomes. Inhibition of miR-155 partly abolished autophagy in N2a cells, which was increased by coculture with miR-155-upregulated exosomes. Mechanistic analysis confirmed that Rheb is a functional target of miR-155 and that microglial exosomal miR-155 accelerated heat stress-induced neuronal autophagy mainly by regulating the Rheb-mTOR signaling pathway.ConclusionIncreased miR-155 in microglial exosomes after heat stroke can induce neuronal autophagy via their transfer into neurons. miR-155 exerted these effects by targeting Rheb, thus inhibiting the activity of mTOR signaling. Therefore, miR-155 could be a promising target for interventions of neuronal autophagy after heat stroke.

Project description:BackgroundProlonged cellular activity may overload cell function, leading to high rates of protein synthesis and accumulation of misfolded or unassembled proteins, which cause endoplasmic reticulum (ER) stress and activate the unfolded protein response (UPR) to re-establish normal protein homeostasis. Previous molecular work has demonstrated that sleep deprivation (SD) leads to ER stress in neurons, with a number of ER-specific proteins being upregulated to maintain optimal cellular proteostasis. It is still not clear which cellular processes activated by sleep deprivation lead to ER- stress, but increased cellular metabolism, higher request for protein synthesis, and over production of oxygen radicals have been proposed as potential contributing factors. Here, we investigate the transcriptional and ultrastructural ER and mitochondrial modifications induced by sleep loss.ResultsWe used gene expression analysis in mouse forebrains to show that SD was associated with significant transcriptional modifications of genes involved in ER stress but also in ER-mitochondria interaction, calcium homeostasis, and mitochondrial respiratory activity. Using electron microscopy, we also showed that SD was associated with a general increase in the density of ER cisternae in pyramidal neurons of the motor cortex. Moreover, ER cisternae established new contact sites with mitochondria, the so-called mitochondria associated membranes (MAMs), important hubs for molecule shuttling, such as calcium and lipids, and for the modulation of ATP production and redox state. Finally, we demonstrated that Drosophila male mutant flies (elav > linker), in which the number of MAMs had been genetically increased, showed a reduction in the amount and consolidation of sleep without alterations in the homeostatic sleep response to SD.ConclusionsWe provide evidence that sleep loss induces ER stress characterized by increased crosstalk between ER and mitochondria. MAMs formation associated with SD could represent a key phenomenon for the modulation of multiple cellular processes that ensure appropriate responses to increased cell metabolism. In addition, MAMs establishment may play a role in the regulation of sleep under baseline conditions.

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Aging results in increased autophagy of mitochondria and protein nitration in rat hepatocytes following heat stress.

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Aging results in increased autophagy of mitochondria and protein nitration in rat hepatocytes following heat stress.

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