Concurrent detection of autolysosome formation and lysosomal degradation by flow cytometry in a high-content screen for inducers of autophagy

<p>Abstract</p> <p>Background</p> <p>Autophagy mediates lysosomal degradation of cytosolic components. Recent work has associated autophagic dysfunction with pathologies, including cancer and cardiovascular disease. To date, the identification of clinically-applicable drugs that modulate autophagy has been hampered by the lack of standardized assays capable of precisely reporting autophagic activity.</p> <p>Results</p> <p>We developed and implemented a high-content, flow-cytometry-based screening approach for rapid, precise, and quantitative measurements of pharmaceutical control over autophagy. Our assay allowed for time-resolved individual measurements of autolysosome formation and degradation, and endolysosomal activities under both basal and activated autophagy conditions. As proof of concept, we analyzed conventional autophagy regulators, including cardioprotective compounds aminoimidazole carboxamide ribonucleotide (AICAR), rapamycin, and resveratrol, and revealed striking conditional dependencies of rapamycin and autophagy inhibitor 3-methyladenine (3-MA). To identify novel autophagy modulators with translational potential, we screened the Prestwick Chemical Library of 1,120 US Food and Drug Administration (FDA)-approved compounds for impact on autolysosome formation. In all, 38 compounds were identified as potential activators, and 36 as potential inhibitors of autophagy. Notably, amongst the autophagy enhancers were cardiac glycosides, from which we selected digoxin, strophanthidin, and digoxigenin for validation by standard biochemical and imaging techniques. We report the induction of autophagic flux by these cardiac glycosides, and the concentrations allowing for specific enhancement of autophagic activities without impact on endolysosomal activities.</p> <p>Conclusions</p> <p>Our systematic analysis of autophagic and endolysosomal activities outperformed conventional autophagy assays and highlights the complexity of drug influence on autophagy. We demonstrate conditional dependencies of established regulators. Moreover, we identified new autophagy regulators and characterized cardiac glycosides as novel potent inducers of autophagic flux.</p

7.胃管アレルギーに於ける腸粘膜スメアーの研究(第二報)(第415回千葉医学会例会,第63回日本小児科学会千葉地方会総会)

Extended representative data set of lysosomal positioning. (a) Schematic representation of the feature “MAX Contour Position” used to quantify lysosomal positioning. (b) Representative LAMP1 immunofluorescence images for different ranges of the feature “LAMP1 MAX Contour Position” in HeLa cells treated as in Fig. 6. (JPG 3911 kb

HUWE1 E3 ligase promotes PINK1/PARKINindependent mitophagy by regulating AMBRA1 activation via IKKa

The selective removal of undesired or damaged mitochondria by autophagy, known as mitophagy, is crucial for cellular homoeostasis, and prevents tumour diffusion, neurodegeneration and ageing. The pro-autophagic molecule AMBRA1 (autophagy/beclin-1 regulator-1) has been defined as a novel regulator of mitophagy in both PINK1/PARKIN-dependent and -independent systems. Here, we identified the E3 ubiquitin ligase HUWE1 as a key inducing factor in AMBRA1-mediated mitophagy, a process that takes place independently of the main mitophagy receptors. Furthermore, we show that mitophagy function of AMBRA1 is post-translationally controlled, upon HUWE1 activity, by a positive phosphorylation on its serine 1014. This modification is mediated by the IKKα kinase and induces structural changes in AMBRA1, thus promoting its interaction with LC3/GABARAP (mATG8) proteins and its mitophagic activity. Altogether, these results demonstrate that AMBRA1 regulates mitophagy through a novel pathway, in which HUWE1 and IKKα are key factors, shedding new lights on the regulation of mitochondrial quality control and homoeostasis in mammalian cells

Myocardial Autophagy after Severe Burn in Rats

Autophagy plays a major role in myocardial ischemia and hypoxia injury. The present study investigated the effects of autophagy on cardiac dysfunction in rats after severe burn.Protein expression of the autophagy markers LC3 and Beclin 1 were determined at 0, 1, 3, 6, and 12 h post-burn in Sprague Dawley rats subjected to 30% total body surface area 3rd degree burns. Autophagic, apoptotic, and oncotic cell death were evaluated in the myocardium at each time point by immunofluorescence. Changes of cardiac function were measured in a Langendorff model of isolated heart at 6 h post-burn, and the autophagic response was measured following activation by Rapamycin and inhibition by 3-methyladenine (3-MA). The angiotensin converting enzyme inhibitor enalaprilat, the angiotensin receptor I blocker losartan, and the reactive oxygen species inhibitor diphenylene iodonium (DPI) were also applied to the ex vivo heart model to examine the roles of these factors in post-burn cardiac function.Autophagic cell death was first observed in the myocardium at 3 h post-burn, occurring in 0.008 ± 0.001% of total cardiomyocytes, and continued to increase to a level of 0.022 ± 0.005% by 12 h post-burn. No autophagic cell death was observed in control hearts. Compared with apoptosis, autophagic cell death occurred earlier and in larger quantities. Rapamycin enhanced autophagy and decreased cardiac function in isolated hearts 6 h post-burn, while 3-MA exerted the opposite response. Enalaprilat, losartan, and DPI all inhibited autophagy and enhanced heart function.Myocardial autophagy is enhanced in severe burns and autophagic cell death occurred early at 3 h post-burn, which may contribute to post-burn cardiac dysfunction. Angiotensin II and reactive oxygen species may play important roles in this process by regulating cell signaling transduction

Lose it or keep it: (how bivalves can provide) insights into mitochondrial inheritance mechanisms

The strictly maternal inheritance (SMI) is a pattern of mitochondrial inheritance observed across the whole animal kingdom. However, some interesting exceptions are known for the class Bivalvia, in which several species show an unusual pattern called doubly uniparental inheritance (DUI) whose outcome is a heteroplasmic pool of mtDNA in males. Even if DUI has been studied for long, its molecular basis has not been established yet. The aim of this work is to select classes of proteins known to be involved in the maintenance of SMI and to compare their features in two clam species differing for their mitochondrial inheritance mechanism, that is, the SMI species Ruditapes decussatus and the DUI species Ruditapes philippinarum. Data have been obtained from the transcriptomes of male and female ripe gonads of both species. Our analysis focused on nucleases and polymerases, ubiquitination and ubiquitin-like modifier pathways, and proteins involved in autophagy and mitophagy. For each protein group of interest, transcription bias (male or female), annotation, and mitochondrial targeting (when appropriate) were assessed. We did not find evidence supporting a role of nucleases/polymerases or autophagic machinery in the enforcement of SMI in R. decussatus. On the other hand, ubiquitinating enzymes with the expected features have been retrieved, providing us with two alternative testable models for mitochondrial inheritance mechanisms at the molecular level

Mutant Huntingtin induces activation of the Bcl-2/adenovirus E1B 19-kDa interacting protein (BNip3)

Huntington's disease (HD) is a neurodegenerative disorder characterized by progressive neuronal death in the basal ganglia and cortex. Although increasing evidence supports a pivotal role of mitochondrial dysfunction in the death of patients' neurons, the molecular bases for mitochondrial impairment have not been elucidated. We provide the first evidence of an abnormal activation of the Bcl-2/adenovirus E1B 19-kDa interacting protein 3 (BNip3) in cells expressing mutant Huntingtin. In this study, we show an abnormal accumulation and dimerization of BNip3 in the mitochondria extracted from human HD muscle cells, HD model cell cultures and brain tissues from HD model mice. Importantly, we have shown that blocking BNip3 expression and dimerization restores normal mitochondrial potential in human HD muscle cells. Our data shed light on the molecular mechanisms underlying mitochondrial dysfunction in HD and point to BNip3 as a new potential target for neuroprotective therapy in HD

Disruption of Neuronal Autophagy by Infected Microglia Results in Neurodegeneration

There is compelling evidence to support the idea that autophagy has a protective function in neurons and its disruption results in neurodegenerative disorders. Neuronal damage is well-documented in the brains of HIV-infected individuals, and evidence of inflammation, oxidative stress, damage to synaptic and dendritic structures, and neuronal loss are present in the brains of those with HIV-associated dementia. We investigated the role of autophagy in microglia-induced neurotoxicity in primary rodent neurons, primate and human models. We demonstrate here that products of simian immunodeficiency virus (SIV)-infected microglia inhibit neuronal autophagy, resulting in decreased neuronal survival. Quantitative analysis of autophagy vacuole numbers in rat primary neurons revealed a striking loss from the processes. Assessment of multiple biochemical markers of autophagic activity confirmed the inhibition of autophagy in neurons. Importantly, autophagy could be induced in neurons through rapamycin treatment, and such treatment conferred significant protection to neurons. Two major mediators of HIV-induced neurotoxicity, tumor necrosis factor-α and glutamate, had similar effects on reducing autophagy in neurons. The mRNA level of p62 was increased in the brain in SIV encephalitis and as well as in brains from individuals with HIV dementia, and abnormal neuronal p62 dot structures immunoreactivity was present and had a similar pattern with abnormal ubiquitinylated proteins. Taken together, these results identify that induction of deficits in autophagy is a significant mechanism for neurodegenerative processes that arise from glial, as opposed to neuronal, sources, and that the maintenance of autophagy may have a pivotal role in neuroprotection in the setting of HIV infection

Multi-Parametric Analysis and Modeling of Relationships between Mitochondrial Morphology and Apoptosis

Mitochondria exist as a network of interconnected organelles undergoing constant fission and fusion. Current approaches to study mitochondrial morphology are limited by low data sampling coupled with manual identification and classification of complex morphological phenotypes. Here we propose an integrated mechanistic and data-driven modeling approach to analyze heterogeneous, quantified datasets and infer relations between mitochondrial morphology and apoptotic events. We initially performed high-content, multi-parametric measurements of mitochondrial morphological, apoptotic, and energetic states by high-resolution imaging of human breast carcinoma MCF-7 cells. Subsequently, decision tree-based analysis was used to automatically classify networked, fragmented, and swollen mitochondrial subpopulations, at the single-cell level and within cell populations. Our results revealed subtle but significant differences in morphology class distributions in response to various apoptotic stimuli. Furthermore, key mitochondrial functional parameters including mitochondrial membrane potential and Bax activation, were measured under matched conditions. Data-driven fuzzy logic modeling was used to explore the non-linear relationships between mitochondrial morphology and apoptotic signaling, combining morphological and functional data as a single model. Modeling results are in accordance with previous studies, where Bax regulates mitochondrial fragmentation, and mitochondrial morphology influences mitochondrial membrane potential. In summary, we established and validated a platform for mitochondrial morphological and functional analysis that can be readily extended with additional datasets. We further discuss the benefits of a flexible systematic approach for elucidating specific and general relationships between mitochondrial morphology and apoptosis

Common Traits Spark the Mitophagy/Xenophagy Interplay

Selective autophagy contributes to the wellbeing of eukaryotic cells by recycling cellular components, disposing damaged organelles, and removing pathogens, amongst others. Both the quality control process of selective mitochondrial autophagy (Mitophagy) and the defensive process of intracellular pathogen-engulfment (Xenophagy) are facilitated via protein assemblies which have shared molecules, a prime example being the Tank-Binding Kinase 1 (TBK1). TBK1 plays a central role in the immunity response driven by Xenophagy and was recently shown to be an amplifying mechanism in Mitophagy, bring to attention the potential cross talk between the two processes. Here we draw parallels between Xenophagy and Mitophagy, speculating on the inhibitory mechanisms of specific proteins (e.g., the 18 kDa protein TSPO), how the preferential sequestering toward one of the two pathways may undermine the other, and in this way impair cellular response to pathogens and cellular immunity. We believe that an in depth understanding of the commonalities may present an opportunity to design novel therapeutic strategies targeted at both the autonomous and non-autonomous processes of selective autophagy