Analog modeling of glucagon-induced autophagy in rat liver. I. Conceptual and mathematical model of telolysosome-autophagosome-autolysosome interaction.
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Animals↗
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Analog modeling of glucagon-induced autophagy in rat liver. II. Evaluation of iron labeling as a means for identifying telolysosome, autophagosome and autolysosome populations.
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Acid Phosphatase↗
[Leydig cells--a normal cell model of cellular autophagy].
In the present study, we tried to estimate, in a semiquantitative way, the relative frequency of the autophagic activity in various cell types under physiological condition. The results indicated that the highest activity appeared to be in the Leydig cells of rat testes. Autophagosomes were frequently observed in electron microscope photographs of Leydig cells, which provide a good model to study the autophagocytosis in normal cells. The autophagic process in Leydig cells was observed with the electron microscope in preparations treated to show CMPase activity. The mode of formation of autophagosomes in Leydig cells can be divided into three steps. Step 1, flattened membranous elements expand to enclose a small cytoplasmic territory to form pre-autophagosome. Step 2, The double membrane profile of the pre-autophagosome then completely encloses the cytoplasmic territory to form early autophagosome in which structurally normal organelles are contained. Step 3, the transformation of an early autophagosome into a late one is accompanied by the loss of one of the two delimiting membranes, the partial disintegration of the enclosed content and simultaneous acquisition of acid phosphatase activity. The enzymatic reactivity is acquired following a close association with the lysosomes. The late autophagosome then reaches the cell surface and appear to exocytose their residual content.
Animals↗
Clinical and biochemical footprints of inherited disorders of autophagy.
Autophagy is an evolutionarily conserved lysosomal recycling system that integrates nutrient sensing, organelle quality control, proteostasis, cellular stress responses and metabolic adaptation. Autophagy is particularly relevant for post-mitotic tissue such as neurons, skin, and immune cells. Monogenic disorders disrupting autophagy or closely coupled endolysosomal trafficking pathways have recently emerged as a recognizable group of inherited metabolic diseases. These conditions are individually rare inborn errors of metabolism and collectively important because they bridge neurodevelopmental, neuromuscular and neurodegenerative disorders, including hereditary forms of Parkinson's disease, spastic paraplegias and neurodegeneration with brain iron accumulation. Multisystem involvement is common but variable. The prototypic disorder is EPG5-related Vici syndrome, in which defective autophagosome-lysosome fusion causes severe neurodevelopmental and multisystem disease. Other disorders may affect any step of the pathway, from phosphatidylinositol 3-phosphate effector biology and ATG conjugation/lipidation to autophagosome maturation, ATG9 trafficking, HOPS/CORVET-related vesicle trafficking (including VPS16 and VPS33A), autophagosome-lysosome fusion, autolysosome reformation and lysosome-mTOR signaling. Clinically, affected individuals commonly present with global developmental delay and/or intellectual disability, epilepsy, movement disorders including dystonia, parkinsonism, ataxia and spasticity, and both neuropathic and myopathic neuromuscular manifestations. A biphasic course with progressive neurodegeneration and variable multisystem (including ocular, cardiac, immunological, cutaneous and growth) involvement are important clinical clues. Diagnosis relies on careful phenotyping, brain MRI, targeted metabolic exclusion of mimics, genomic sequencing and functional assays in patient-derived cells as required. Supportive multidisciplinary management is essential. No disease-modifying therapy is currently established in humans, but pathway-based cellular assays, model systems and small-molecule or gene-replacement strategies are creating a rational therapeutic pipeline. Importantly, IEMbase dyadic nomenclature with system-level clinical annotations provides a standardized framework for quantifying shared phenotypic signatures across these ultra-rare conditions. This review summarizes pathobiochemistry, genetics, clinical presentation, diagnosis and treatment prospects for inherited disorders of autophagy.
Autophagosome↗
The pathway of autophagy in the epigenetic landscape of Mycobacterium-host interactions.
Macroautophagy (autophagy) is an evolutionarily conserved process that degrades excess cytoplasmic components, such as protein aggregates and damaged organelles, by encapsulating them within double-membrane autophagosomes. These autophagosomes undergo distinct stages - initiation, phagophore nucleation, expansion, and closure - before fusing with lysosomes (or occasionally endosomes) for degradation and recycling. This process is regulated by ATG (autophagy related) proteins, which govern autophagosome formation and lysosomal fusion. Epigenetic modifications and transcription factors can regulate ATG gene expression in the nucleus. Autophagy also plays a key role in eliminating intracellular Mycobacterium tuberculosis (Mtb) through the lytic and antimicrobial activities of autolysosomes, which are more potent antimicrobial compartments than conventional phagosomes. Emerging evidence suggests that Mtb can modify the host epigenome and transcriptional machinery, significantly affecting the host immune response. This review explores the epigenetic regulation of autophagy during mycobacterium-host interactions. The interplay between epigenetic regulation and autophagy highlights a crucial aspect of host-pathogen interactions during Mtb infection. Understanding how Mtb manipulates the host epigenome to regulate autophagy could lead to the development of novel therapeutic strategies that enhance autophagic pathways or counteract Mtb's immune evasion tactics.Abbreviations: AM: Alveolar macrophages; ATG: autophagy related; DNMT: DNA methyltransferase; FOXO3: forkhead box O3; HAT: histone acetyltransferase; HDAC: histone deacetylase; MIR: microRNA; MTOR: mechanistic target of rapamycin kinase; Mtb: Mycobacterium tuberculosis; ROS: reactive oxygen species; SIRT: sirtuin; STPK: serine/threonine protein kinase.
Autophagy↗
Cytoskeletal elements are required for the formation and maturation of autophagic vacuoles.
We evaluated the role of cytoskeletal elements in the degradation of endogenous proteins via autophagy using biochemical and morphological techniques. In the absence of exogenous amino acids, degradation of endogenous proteins was enhanced in cultured normal rat kidney cells. This enhanced degradative state was accompanied by a 4-fold increase in the occurrence of autophagic vacuoles. In the presence of drugs that induce the depolymerization of microfilaments (cytochalasins B and D) or microtubules (nocodazole), protein degradation was not enhanced in nutrient-deprived cells. Although these drugs had similar inhibitory effects on the protein degradation, their effect on autophagy differed. Cytochalasins B and D interfered with the formation of the autophagosome. In cells treated with these drugs, the fractional volume represented by autophagic vacuoles was not substantially increased despite nutrient depletion. On the contrary, nocodazole appeared to have no effect on the formation of autophagosomes. Instead, this drug suppressed the delivery of hydrolytic enzymes, thereby resulting in an accumulation of acidic autophagic vacuoles containing undegraded cellular components.
Actin Cytoskeleton↗
Bi-allelic ATG4D variants are associated with a neurodevelopmental disorder characterized by speech and motor impairment.
Autophagy regulates the degradation of damaged organelles and protein aggregates, and is critical for neuronal development, homeostasis, and maintenance, yet few neurodevelopmental disorders have been associated with pathogenic variants in genes encoding autophagy-related proteins. We report three individuals from two unrelated families with a neurodevelopmental disorder characterized by speech and motor impairment, and similar facial characteristics. Rare, conserved, bi-allelic variants were identified in ATG4D, encoding one of four ATG4 cysteine proteases important for autophagosome biogenesis, a hallmark of autophagy. Autophagosome biogenesis and induction of autophagy were intact in cells from affected individuals. However, studies evaluating the predominant substrate of ATG4D, GABARAPL1, demonstrated that three of the four ATG4D patient variants functionally impair ATG4D activity. GABARAPL1 is cleaved or "primed" by ATG4D and an in vitro GABARAPL1 priming assay revealed decreased priming activity for three of the four ATG4D variants. Furthermore, a rescue experiment performed in an ATG4 tetra knockout cell line, in which all four ATG4 isoforms were knocked out by gene editing, showed decreased GABARAPL1 priming activity for the two ATG4D missense variants located in the cysteine protease domain required for priming, suggesting that these variants impair the function of ATG4D. The clinical, bioinformatic, and functional data suggest that bi-allelic loss-of-function variants in ATG4D contribute to the pathogenesis of this syndromic neurodevelopmental disorder.
Journal Article↗
Character and origin of vacuoles induced in mammalian cells by the cytotoxin of Helicobacter pylori.
Cytotoxic activity of culture supernates of Helicobacter pylori is manifested by vacuolation of mammalian cells in vitro. The formation and maturation of toxin-induced vacuoles in HeLa cells has been studied to examine the possibility that they are autophagosomal in nature. Observation by light microscopy revealed that vacuoles originate in a perinuclear position, increasing in number and size until cell degeneration and lysis occur after 48 h. Ultrastructural study of mature vacuoles indicated the presence of a bounding membrane with contents consisting of degenerate cytoplasmic components and acid phosphatase activity. Confocal fluorescence imaging demonstrated acridine orange accumulation in the vacuoles of toxin-treated cells, indicating an acidic intravacuolar pH. These features are characteristic of autophagosomes. In addition, the size of vacuoles in living, acridine orange-stained cells tended to be inversely proportional to fluorescence intensity. Fluid phase endocytic markers were observed only rarely within nascent vacuoles. Over the succeeding 24 h, labelling of most vacuoles with these dyes was observed. This, along with the observation of intravacuolar acid phosphatase activity, provides evidence that vacuoles communicate at some point during their development with endocytically derived compartments. These observations provide direct evidence for an autophagic origin of these structures.
Acid Phosphatase↗
Does chloroquine, an antimalarial drug, affect autophagy in Tetrahymena pyriformis?
The effect of chloroquine (CQ) on autophagy was studied in starved Tetrahymena pyriformis. When a proliferating Tetrahymena culture is transferred to a starvation medium, autophagy commences although cells most advanced in the cell cycle will divide. The drug was added to 1-h starved cells at different pH values because CQ affects pH dependently. The CQ concentration blocking all cell divisions was determined as the lowest toxic, but sublethal, concentration. Hence, the highest tolerated concentrations at pH 6.8, 7.1, and 7.7 were 1.0, 0.3, and 0.03 mM CQ, respectively. Lower CQ concentrations had a dose-dependent effect on cell increment and higher concentrations induced cell mortality. Rates of cell motility and decreases in cell volume were affected by the drug, while the capacity for endocytosis was unaffected in low concentrations but affected dose dependently in high concentrations. Light microscopically, all drug-treated cells contained small refractive bodies, but in toxic concentrations they also contained conspicuously large vacuoles. After 1 h and 4 h in CQ, fine structure analysis showed autophagosomes with electron-dense material in cells in tolerated concentrations and of enlarged size, but decreased number, in toxic concentrations. The contents of autophagosomes revealed cell organelles in different stages of disintegration. The conclusion is that the drug enhances autophagy in Tetrahymena in a pH-, dose-, and time-dependent manner.
Animals↗
A subcomplex comprising TRAPPC11, TRAPPC12, TRAPPC13 and the fungal TRAPPC2L homolog, Tca17, directs TRAPPIII to autophagy.
Transport protein particle complexes (TRAPPs) are master regulators of membrane trafficking. TRAPPs are targeted to different locales by pathway-specific subunits decorating a core hetero-heptamer to build TRAPPII (Golgi exit) and TRAPPIII (autophagosomes and ER-Golgi trafficking). Metazoan and Arabidopsis TRAPPIII have three components, TRAPPC11, TRAPPC12 and TRAPPC13 (hereafter denoted TRAPPC11/12/13), that are absent from budding yeast. We studied TRAPPC11/12/13 in the related ascomycete Aspergillus nidulans, where TRAPPC11 and TRAPPC12 localize to pre-autophagosomes and their ablation impairs autophagy. We found that two stable subcomplexes containing Tca17, the homolog of metazoan TRAPPC2L, coexist - one includes the TRAPPII-specific subunits Trs120, Trs130 and Trs65 whereas the other contains the TRAPPIII-specific subunits TRAPPC11/12/13. Both are recruited to core TRAPP by Tca17, which therefore plays a crucial role by determining the physiological role of TRAPP. TRAPPIII also exists in two versions, TRAPPIIIa and TRAPPIIIb, both of which contain Trs85, the homolog of metazoan TRAPPC8, but with only TRAPPIIIb containing TRAPPC11/12/13, which target TRAPPIII to autophagy. This study might help characterize potentially pathogenic mutations affecting human TRAPPC11/12/13, facilitating assessment of their functional consequences in a genetically amenable ascomycete.
Autophagy↗
Fine structure of the small intestinal mucosa in infantile marasmic malnutrition.
The jejunal mucosa was studied in infantile marasmic malnutrition in the early phase after treatment was begun and before the onset of significant weight gain. In 7 infants light microscopy before recovery showed that the mucosa was normal or mildly abnormal in 4, and moderately abnormal in 3 cases. The electron microscope disclosed abnormalities of the brush border, large autophagosomes and residual bodies, and the deposition of collagen, filaments, and a dense, finely granular material below the basal lamella. Three of the infants were studied again during recovery. Although the histology remained unchanged, electron microscopy revealed improvement of the brush border, disappearance of the autophagosomes, and smaller and fewer residual bodies. The dense material below the basal lamella was absent whereas the fibrillar components remained. It is postulated that the fine structural lesions observed may be due to the derangements in cell metabolism caused by the severe, prolonged restriction of protein and caloric intake.
Cell Membrane↗
Panduratin A Induces Autophagy Through AMPK Activation Independent of mTOR Inhibition and Restricts Mycobacterium tuberculosis in Host Macrophages.
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a major global health burden, especially with the increasing prevalence of drug-resistant strains. There is an urgent need for new therapeutics that act via alternative mechanisms. Autophagy, a vital cell-autonomous defense process, allows macrophages to degrade intracellular pathogens such as Mtb and has gained attention as a potential target for host-directed therapy. In this study, we conducted a high-content imaging screen of herb-derived compounds to identify autophagy inducers in RAW264.7 macrophages. Panduratin A (NPA), a natural compound from Boesenbergia rotunda, was found to potently induce autophagy. NPA promoted autophagic vacuole formation in a dose-dependent fashion at low micromolar levels. Its autophagy-inducing effect was validated using RFP-GFP-LC3 dual fluorescence assays and immunoblotting in the presence of bafilomycin A1. Further mechanistic analysis revealed that NPA activates autophagy through AMPK activation, independent of mTOR inhibition. Importantly, NPA significantly promoted intracellular Mtb clearance and increased colocalization of Mtb with autophagosomes and lysosomes, in a manner dependent on Beclin-1. These findings highlight NPA as a potent enhancer of macrophage antimicrobial responses via autophagy, supporting its potential as a candidate for host-directed adjunctive therapy against TB.
Autophagy↗
The pathogenesis of lupinosis in the rat.
The morphological, histochemical and biochemical changes in the liver of rats challenged with a single intraperitoneal injection of the toxin of lupinosis were investigated. The resulting injury is biphasic. The initial non-specific phase lasts for a few days, affects mainly the centrilobular region and is characterised by mitochondrial swelling, lipid accummulation and autophagosome formation. In addition, the hepatic concentrations of OCT, LDH, SDH, and AST fall while that for ALP increase. This is accompanied by a rise in the serum concentration of many of these enzymes. The second phase lasts longer and is characterised by phenomena indicative of abnormalities in hepatocytic division. Normal and abnormal metaphase as well as multinucleated hepatocytes falls. The possibility that the toxin of lupinosis interferes with microtubular units is considered.
Animals↗
Association of myocardial cell necrosis with experimental cardiac hypertrophy.
Cardiac hypertrophy was induced in rabbits by injecting thyroxine or isoprenaline, or by surgically constricting the abdominal aorta. An increase in heart weight was associated with a change in the ratios of bound to free forms of five lysosomal enzymes, a change in serum creatine phosphokinase and lactate dehydrogenase, and a change in the morphology of the myocardial cells. Isoprenaline treatment for 5 days induced a maximal change in heart weight, in the ratio of lysosomal enzymes, and in the serum enzymes. Thyroxine treatment was required for 15 days before maximal changes in heart weight, ratio, and serum enzymes were observed. In contrast, coarctation of the aorta caused a progressive change in heart weight, in the ratio of lysosomal enzymes, and in serum enzymes. These results suggest that necrosis of the myocardial cells does indeed accompany cardiac hypertrophy. It was further observed that autophagosomes, degenerating mitochondria in the myocardial cells during the induction of cardiac hypertrophy, and myofibril lysis were found, all of which confirms the suggestion of myocardial cell necrosis in the experimentally enlarged heart.
Animals↗
Effects of antimicrotubular agents on the fine structure of the Golgi complex in embryonic chick osteoblasts.
Embryonic chick frontal bones were cultured in the presence of colchicine or vinblastine and subsequently examined by tranmission electron microscopy. In control cultures the osteoblasts showed a large Golgi complex consisting of dictyosomes arranged in a well-defined juxtanuclear area. Microtubules were particularly numerous within this Golgi area although they could be observed throughout the cytoplasm. Colchicine and vinblastine caused the disappearance of cytoplasmic microtubules, while bundles of 10 nm diameter filaments appeared more frequently. In addition, cell polarity was lost and the Golgi complex became disorganized, with the dictyosomes randomly dispersed in the cytoplasm and showing a decreased number of cisternae and an increased number of vacuoles, the latter generally lacking stainable material. Increased number of autophagosomes were also noted. These findings indicate that microtubules function in the organization of the Golgi complex in osteoblasts. In view of the well documented role of this organelle system in collagen secretion it is suggested that previously observed secretory disturbances produced by antimicrotubular drugs may be due to a defective transfer of material to the dictyosomes and/or a defect in the packaging and transport of such material away from them.
Animals↗
Synthesis, secretion and immunoelectron microscopic demonstration of apolipoprotein B-containing lipoprotein particles in the visceral rat yolk sac.
Electron microscopic investigations on the involvement of the fetal membranes of the rat (visceral yolk sac) in the lipid metabolism revealed the occurrence of lipoprotein-sized particles located in cisternal Golgi stacks, Golgi vesicles and secretory vesicles of the cells of the visceral yolk sac epithelium as well as in distended areas of the intercellular space between adjacent epithelial cells. Application of the protein A-gold technique with specific anti-apoB antiserum resulted in a specific location of immunogold both over the different compartments of the lipoprotein pathway (RER, Golgi complex, secretory vesicles) as well as over the distended intercellular spaces, thus confirming these particles to be lipoproteins in nature. Isolated visceral epithelial cells prepared by a tryptic digestion method exhibited some ultrastructural alterations, such as a loss of apical brush border, a change from columnar to spherical cell shape, a decrease in phagolysosomes, but an increase in autophagosomal structures after 6 h incubation at a vitality rate of at least 85%. Within this period the epithelial cells secreted measurable amounts of apoB-containing lipoproteins into the medium floating in the density classes d less than 1.006 g/ml, d = 1.006-1.020 g/ml and d = 1.020-1.064 g/ml. The production of the lipoproteins was partly inhibited by cycloheximide indicating the secretion of particles with performed as well as newly synthesized apoB. Negative staining of the particles revealed an average diameter of 34 nm of VLDL, 31 nm of IDL and 24 nm of LDL. In summary, our studies demonstrate that in the feto-placental unit of the rat the fetal membranes are capable of synthesizing and secreting lipoproteins. The cells of the visceral yolk sac epithelium were shown to be the producers of apoB-containing particles.
Animals↗
Reexamination of granulovacuolar degeneration.
Granulovacuolar degeneration (GVD) in the hippocampal pyramidal neurons of Alzheimer-type dementia was examined. Immunohistochemical examinations showed that the majority of centrally located granules were positive for ubiquitin. Based on electron microscopic observations, morphogenesis of GVD is considered to be as follows. Slight-to-moderate amounts of electron-dense material appear in the cytoplasm at the early stage, and are then surrounded and demarcated by a two-layered membrane (probably from smooth endoplasmic reticulum). Following this some inner material is digested forming floccular and liquid-like materials, while undigested material remains as coarse electron-dense granules. Specifically, granulovacuoles are considered to be an age-related special type of autophagosome. Analytical electron microscopy disclosed that the granules in GVD contained some aluminum.
Alzheimer Disease↗
[Histochemical and electron microscopical changes in the kidney of pregnant rats at the time of partus (author's transl)].
In the convoluted proximal tubules of the kidney of pregnant rats alkaline phosphatase activity decreases from 21 days gestation till the 2nd day after birth. During the same time the reaction product of the histochemical succinate dehydrogenase assay exhibits an atypical granular character in some of the proximal tubules, which sometimes additionally contain sudanophilic structures. Electron microscopy reveals dilated mitochondria, an increased number of autophagosomes and big lipid vacuoles of low density.
Alkaline Phosphatase↗