Brain-derived autophagosome profiling reveals the engulfment of nucleoid-enriched mitochondrial fragments by basal autophagy in neurons

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4-hydroxynonenal contributes to NGF withdrawal-induced neuronal apoptosis

Reactive oxygen species are a necessary triggering event for apoptosis of sympathetic neurons after nerve growth factor (NGF) withdrawal. Reactive oxygen species can lead to the generation of 4-hydroxynonenal (HNE), a highly reactive aldehyde that forms adducts with proteins. This covalent modification can activate or inhibit signal transduction pathways involved in the induction of apoptosis. This process may be clinically relevant because HNE-adduct immunoreactivity increases in several disease states. Here we evaluate the role of HNE-adducts in sympathetic neurons undergoing NGF-deprivation-induced apoptosis, a model of developmental programmed cell death. We show that HNE-adduct immunoreactivity is dramatically increased after NGF-withdrawal in an NADPH oxidase-dependent manner. Moreover, HNE-adducts appear to contribute to NGF-deprivation-induced apoptotic signal transduction because microinjected HNE-adduct antiserum protects sympathetic neurons from NGF withdrawal. In conclusion, this report suggests the direct contribution of endogenously generated HNE in the stimulation of apoptotic signal transduction in neurons.     

c-Jun N-terminal kinase regulates mitochondrial bioenergetics by modulating pyruvate dehydrogenase activity in primary cortical neurons

This study examines the role of c- jun N-terminal kinase (JNK) in mitochondrial signaling and bioenergetics in primary cortical neurons and isolated rat brain mitochondria. Exposure of neurons to either anisomycin (an activator of JNK/p38 mitogen-activated protein kinases) or H₂O₂ resulted in activation (phosphorylation) of JNK (mostly p46^JNK1) and its translocation to mitochondria. Experiments with mitochondria isolated from either rat brain or primary cortical neurons and incubated with proteinase K revealed that phosphorylated JNK was associated with the outer mitochondrial membrane; this association resulted in the phosphorylation of the E_1α subunit of pyruvate dehydrogenase, a key enzyme that catalyzes the oxidative decarboxylation of pyruvate and that links two major metabolic pathways: glycolysis and the tricarboxylic acid cycle. JNK-mediated phosphorylation of pyruvate dehydrogenase was not observed in experiments carried out with mitoplasts, thus suggesting the requirement of intact, functional mitochondria for this effect. JNK-mediated phosphorylation of pyruvate dehydrogenase was associated with a decline in its activity and, consequently, a shift to anaerobic pyruvate metabolism: the latter was confirmed by increased accumulation of lactic acid and decreased overall energy production (ATP levels). Pyruvate dehydrogenase appears to be a specific phosphorylation target for JNK, for other kinases, such as protein kinase A and protein kinase C did not elicit pyruvate dehydrogenase phosphorylation and did not decrease the activity of the complex. These results suggest that JNK mediates a signaling pathway that regulates metabolic functions in mitochondria as part of a network that coordinates cytosolic and mitochondrial processes relevant for cell function.     

Time-dependent dysregulation of autophagy: Implications in aging and mitochondrial homeostasis in the kidney proximal tubule

Autophagy plays an essential role in cellular homeostasis through the quality control of proteins and organelles. Although a time-dependent decline in autophagic activity is believed to be involved in the aging process, the issue remains controversial. We previously demonstrated that autophagy maintains proximal tubular cell homeostasis and protects against kidney injury. Here, we extend that study and examine how autophagy is involved in kidney aging. Unexpectedly, the basal autophagic activity was higher in the aged kidney than that in young kidney; short-term cessation of autophagy in tamoxifen-inducible proximal tubule-specific autophagy-deficient mice increased the accumulation of SQSTM1/p62- and ubiquitin-positive aggregates in the aged kidney. By contrast, autophagic flux in response to metabolic stress was blunted with aging, as demonstrated by the observation that transgenic mice expressing a green fluorescent protein (GFP)-microtubule-associated protein 1 light chain 3B fusion construct, showed a drastic increase of GFP-positive puncta in response to starvation in young mice compared to a slight increase observed in aged mice. Finally, proximal tubule-specific autophagy-deficient mice at 24 mo of age exhibited a significant deterioration in kidney function and fibrosis concomitant with mitochondrial dysfunction as well as mitochondrial DNA abnormalities and nuclear DNA damage, all of which are hallmark characteristics of cellular senescence. These results suggest that age-dependent high basal autophagy plays a crucial role in counteracting kidney aging through mitochondrial quality control. Furthermore, a reduced capacity for upregulation of autophagic flux in response to metabolic stress may be associated with age-related kidney diseases.     

The mechanical effects of CRT promoting autophagy via mitochondrial calcium uniporter down‐regulation and mitochondrial dynamics alteration

The mechanism of cardiac resynchronization therapy (CRT) remains unclear. In this study, mitochondria calcium uniporter (MCU), dynamin‐related protein‐1 (DNM1L/Drp1) and their relationship with autophagy in heart failure (HF) and CRT are investigated. Thirteen male beagle's dogs were divided into three groups (sham, HF, CRT). Animals received left bundle branch (LBB) ablation followed by either 8‐week rapid atrial pacing or 4‐week rapid atrial pacing and 4‐week biventricular pacing. Cardiac function was evaluated by echocardiography. Differentially expressed genes (DEGs) were detected by microarray analysis. General morphological changes, mitochondrial ultrastructure, autophagosomes and mitophagosomes were investigated. The cardiomyocyte stretching was adopted to imitate the mechanical effect of CRT. Cells were divided into three groups (control, angiotensin‐II and angiotensin‐II + stretching). MCU, DNM1L/Drp1 and autophagy markers were detected by western blots or immunofluorescence. In the present study, CRT could correct cardiac dysfunction, decrease cardiomyocyte's size, alleviate cardiac fibrosis, promote the formation of autophagosome and mitigate mitochondrial injury. CRT significantly influenced gene expression profile, especially down‐regulating MCU and up‐regulating DNM1L/Drp1. Cell stretching reversed the angiotensin‐II induced changes of MCU and DNM1L/Drp1 and partly restored autophagy. CRT's mechanical effects down‐regulated MCU, up‐regulated DNM1L/Drp1 and subsequently enhanced autophagy. Besides, the mechanical stretching prevented the angiotensin‐II‐induced cellular enlargement.     

Inhibition of glycolysis attenuates 4-hydroxynonenal-dependent autophagy and exacerbates apoptosis in differentiated SH-SY5Y neuroblastoma cells

How cellular metabolic activities regulate autophagy and determine the susceptibility to oxidative stress and ultimately cell death in neuronal cells is not well understood. An important example of oxidative stress is 4-hydroxynonenal (HNE), which is a lipid peroxidation product that is formed during oxidative stress, and accumulates in neurodegenerative diseases causing damage. The accumulation of toxic oxidation products such as HNE, is a prevalent feature of neurodegenerative diseases, and can promote organelle and protein damage leading to induction of autophagy. In this study, we used differentiated SH-SY5Y neuroblastoma cells to investigate the mechanisms and regulation of cellular susceptibility to HNE toxicity and the relationship to cellular metabolism. We found that autophagy is immediately stimulated by HNE at a sublethal concentration. Within the same time frame, HNE induces concentration dependent CASP3/caspase 3 activation and cell death. Interestingly, both basal and HNE-activated autophagy, were regulated by glucose metabolism. Inhibition of glucose metabolism by 2-deoxyglucose (2DG), at a concentration that inhibited autophagic flux, further exacerbated CASP3 activation and cell death in response to HNE. Cell death was attenuated by the pan-caspase inhibitor Z-VAD-FMK. Specific inhibition of glycolysis using koningic acid, a GAPDH inhibitor, inhibited autophagic flux and exacerbated HNE-induced cell death similarly to 2DG. The effects of 2DG on autophagy and HNE-induced cell death could not be reversed by addition of mannose, suggesting an ER stress-independent mechanism. 2DG decreased LAMP1 and increased BCL2 levels suggesting that its effects on autophagy may be mediated by more than one mechanism. Furthermore, 2DG decreased cellular ATP, and 2DG and HNE combined treatment decreased mitochondrial membrane potential. We conclude that glucose-dependent autophagy serves as a protective mechanism in response to HNE.     

Neuronal mitochondrial dynamics coordinate systemic mitochondrial morphology and stress response to confer pathogen resistance in C. elegans

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Involvement of autophagy and mitochondrial dynamics in determining the fate and effects of irreparable mitochondrial DNA damage

Mitochondrial DNA (mtDNA) is different in many ways from nuclear DNA. A key difference is that certain types of DNA damage are not repaired in the mitochondrial genome. What, then, is the fate of such damage? What are the effects? Both questions are important from a health perspective because irreparable mtDNA damage is caused by many common environmental stressors including ultraviolet C radiation (UVC). We found that UVC-induced mtDNA damage is removed slowly in the nematode Caenorhabditis elegans via a mechanism dependent on mitochondrial fusion, fission, and autophagy. However, knockdown or knockout of genes involved in these processes—many of which have homologs involved in human mitochondrial diseases—had very different effects on the organismal response to UVC. Reduced mitochondrial fission and autophagy caused no or small effects, while reduced mitochondrial fusion had dramatic effects.     

Regulation of mitochondrial bioenergetics by the non-canonical roles of mitochondrial dynamics proteins in the heart

Recent advancement in mitochondrial research has significantly extended our knowledge on the role and regulation of mitochondria in health and disease. One important breakthrough is the delineation of how mitochondrial morphological changes, termed mitochondrial dynamics, are coupled to the bioenergetics and signaling functions of mitochondria. In general, it is believed that fusion leads to an increased mitochondrial respiration efficiency and resistance to stress-induced dysfunction while fission does the contrary. This concept seems not applicable to adult cardiomyocytes. The mitochondria in adult cardiomyocytes exhibit fragmented morphology (tilted towards fission) and show less networking and movement as compared to other cell types. However, being the most energy-demanding cells, cardiomyocytes in the adult heart possess vast number of mitochondria, high level of energy flow, and abundant mitochondrial dynamics proteins. This apparent discrepancy could be explained by recently identified new functions of the mitochondrial dynamics proteins. These “non-canonical” roles of mitochondrial dynamics proteins range from controlling inter-organelle communication to regulating cell viability and survival under metabolic stresses. Here, we summarize the newly identified non-canonical roles of mitochondrial dynamics proteins. We focus on how these fission and fusion independent roles of dynamics proteins regulate mitochondrial bioenergetics. We also discuss potential molecular mechanisms, unique intracellular location, and the cardiovascular disease relevance of these non-canonical roles of the dynamics proteins. We propose that future studies are warranted to differentiate the canonical and non-canonical roles of dynamics proteins and to identify new approaches for the treatment of heart diseases. This article is part of a Special issue entitled Cardiac adaptations to obesity, diabetes and insulin resistance, edited by Professors Jan F.C. Glatz, Jason R.B. Dyck and Christine Des Rosiers.     

The Effect of the Lipid Peroxidation Product 4-Hydroxynonenal and of its Metabolite 4-Hydroxynonenoic Acid on Respiration of Rat Kidney Cortex Mitochondria

In rat kidney cortex mitochondria, 4-hydroxynonenal inhibits state 3 respiration as well as uncoupled respiration at micromolar concentrations. The inhibition is more distinct for NAD-linked than for FAD-linked respiration. 4-Hydroxynonenal increases the state 4 respiration. It is assumed that 4-hydroxynonenal behaves like a decoupling agent. 4-Hydroxynonenal augments the inhibitory effect of 2, 4-dinitrophenol observed at superoptimal concentrations. 4-Hydroxynonenal is metabolised by renal mitochondria, and 4-hydroxynonenoic acid is one of the metabolites generated. This metabolite is without effect on respiration at concentrations up to 50 μM. Therefore, the effect of 4-hydroxynonenal on respiration is not mediated by this fatty acid derivative formed during respiratory measurements.     

Increased mitochondrial fusion and autophagy help isolated hepatocytes repolarize in collagen sandwich cultures

Freshly isolated, depolarized rat hepatocytes can repolarize into bile canalicular networks when plated in collagen sandwich cultures. We studied the events underlying this repolarization process, focusing on how hepatocytes restore ATP synthesis and resupply biosynthetic precursors after the stress of being isolated from liver. We found that soon after being plated in collagen sandwich cultures, hepatocytes converted their mitochondria into highly fused networks. This occurred through a combination of upregulation of mitochondrial fusion proteins and downregulation of a mitochondrial fission protein. Mitochondria also became more active for oxidative phosphorylation, leading to overall increased ATP levels within cells. We further observed that autophagy was upregulated in the repolarizing hepatocytes. Boosted autophagy levels likely served to recycle cellular precursors, supplying building blocks for repolarization. Repolarizing hepatocytes also extensively degraded lipid droplets, whose fatty acids provide precursors for β-oxidation to fuel oxidative phosphorylation in mitochondria. Thus, through coordination of mitochondrial fusion, autophagy, and lipid droplet consumption, depolarized hepatocytes are able to boost ATP synthesis and biosynthetic precursors to efficiently repolarize in collagen sandwich cultures.     

MitoTimer probe reveals the impact of autophagy,fusion, and motility on subcellular distribution of young and old mitochondrial protein and on relative mitochondrial protein age

To study mitochondrial protein age dynamics, we targeted a time-sensitive fluorescent protein, MitoTimer, to the mitochondrial matrix. Mitochondrial age was revealed by the integrated portions of young (green) and old (red) MitoTimer protein. Mitochondrial protein age was dependent on turnover rates as pulsed synthesis, decreased import, or autophagic inhibition all increased the proportion of aged MitoTimer protein. Mitochondrial fusion promotes the distribution of young mitochondrial protein across the mitochondrial network as cells lacking essential fusion genes Mfn1 and Mfn2 displayed increased heterogeneity in mitochondrial protein age. Experiments in hippocampal neurons illustrate that the distribution of older and younger mitochondrial protein within the cell is determined by subcellular spatial organization and compartmentalization of mitochondria into neurites and soma. This effect was altered by overexpression of mitochondrial transport protein, RHOT1/MIRO1. Collectively our data show that distribution of young and old protein in the mitochondrial network is dependent on turnover, fusion, and transport.     

Involvement of autophagy and mitochondrial dynamics in determining the fate and effects of irreparable mitochondrial DNA damage

Mitochondrial DNA (mtDNA) is different in many ways from nuclear DNA. A key difference is that certain types of DNA damage are not repaired in the mitochondrial genome. What, then, is the fate of such damage? What are the effects? Both questions are important from a health perspective because irreparable mtDNA damage is caused by many common environmental stressors including ultraviolet C radiation (UVC). We found that UVC-induced mtDNA damage is removed slowly in the nematode Caenorhabditis elegans via a mechanism dependent on mitochondrial fusion, fission, and autophagy. However, knockdown or knockout of genes involved in these processes—many of which have homologs involved in human mitochondrial diseases—had very different effects on the organismal response to UVC. Reduced mitochondrial fission and autophagy caused no or small effects, while reduced mitochondrial fusion had dramatic effects.     

NOX4 regulates autophagy during energy deprivation

NADPH oxidase is a cellular enzyme devoted to the production of reactive oxygen species (ROS). NOX4 and NOX2 are the main isoforms of NADPH oxidase in the cardiovascular system. In our recent study, we demonstrated that NOX4, but not NOX2, is a critical mediator of the cardiomyocyte adaptive response to energy stress. NOX4 activity and protein levels are increased in the endoplasmic reticulum (ER) but not in mitochondria of cardiomyocytes during the early phase of energy deprivation. NOX4-derived production of ROS in the ER is a critical event that activates autophagy through stimulation of the EIF2AK3/PERK-EIF2S1/eIF-2α-ATF4 pathway. NOX4-dependent autophagy is an important mechanism to preserve cellular energy and limit cell death in energy-deprived cardiomyocytes. Aside from elucidating a crucial physiological function of NOX4 during cellular energy stress, our study dissects a novel signaling mechanism that regulates autophagy under this condition.     

Oxidative inactivation of the proteasome in Alzheimer's disease

In the present study we isolated proteasome complexes from control, mild cognitive impairment (MCI), and Alzheimer's disease (AD) subjects. No significant difference in the amount of proteasomes was detected across the different groups, although impairments in chymotrypsin-like proteasome activity was observed in AD subjects. Large impairments in proteasome- mediated degradation of an oxidized protein substrate was observed in MCI and AD subjects. Incubation with a reducing agent (DTT) had no significant effect on proteasome chymotrypsin-like activity, but fully restored proteasome-mediated protein degradation in MCI and AD subjects. Proteasomes from AD subjects exhibited elevations in protein carbonyls, 4-hydroxynonenal-conjugation, and neuroprostane-conjugation. Together, these data confirm that impairments in the function of purified proteasomes occurs in the earliest stages of AD, and directly support a role for oxidative inactivation contributing to declines in proteasome function in AD.