Inhibiting autophagy targets human leukemic stem cells and hypoxic AML blasts by disrupting mitochondrial homeostasis.

Leukemia stem cells (LSCs) and therapy-resistant acute myeloid leukemia (AML) blasts contribute to the reinitiation of leukemia after remission, necessitating therapeutic interventions that target these populations. Autophagy is a prosurvival process that allows for cells to adapt to a variety of stressors. Blocking autophagy pharmacologically by using mechanistically distinct inhibitors induced apoptosis and prevented colony formation in primary human AML cells. The most effective inhibitor, bafilomycin A1 (Baf A1), also prevented the in vivo maintenance of AML LSCs in NSG mice. To understand why Baf A1 exerted the most dramatic effects on LSC survival, we evaluated mitochondrial function. Baf A1 reduced mitochondrial respiration and stabilized PTEN-induced kinase-1 (PINK-1), which initiates autophagy of mitochondria (mitophagy). Interestingly, with the autophagy inhibitor chloroquine, levels of enhanced cell death and reduced mitochondrial respiration phenocopied the effects of Baf A1 only when cultured in hypoxic conditions that mimic the marrow microenvironment (1% O2). This indicates that increased efficacy of autophagy inhibitors in inducing AML cell death can be achieved by concurrently inducing mitochondrial damage and mitophagy (pharmacologically or by hypoxic induction) and blocking mitochondrial degradation. In addition, prolonged exposure of AML cells to hypoxia induced autophagic flux and reduced chemosensitivity to cytarabine (Ara-C), which was reversed by autophagy inhibition. The combination of Ara-C with Baf A1 also decreased tumor burden in vivo. These findings demonstrate that autophagy is critical for mitochondrial homeostasis and survival of AML cells in hypoxia and support the development of autophagy inhibitors as novel therapeutic agents for AML.

A case of multiple recurrent intussusceptions due to multiple lymphomatous polyposis associated with diffuse large B-cell lymphoma of gastrointestinal tract in a 15-year-old child: A rare case report.

INTRODUCTION AND IMPORTANCE: Multiple lymphomatous polyposis (MLP) is a distinctive and rare entity of primary gastrointestinal (GI) lymphoma characterized by polypoid lymphomatous tissue in long segments of the gut and a strong tendency for spread throughout the GI tract. Although many cases of MLP presenting as intussusceptions in adults have been reported, we report a rare case of multiple recurrent intussusceptions due to MLP associated with high-grade Diffuse Large B-cell lymphoma (DLBCL) of the entire GI tract in a 15-year-old child. CASE PRESENTATION: A 15-year-old child previously operated for acute intestinal obstruction, presented with intermittent abdominal pain, nausea and vomiting. Imaging studies confirmed the diagnosis of multiple small bowel intussusceptions. Patient was treated by exploratory laparotomy and multiple resection anastomosis. Histopathology confirmed the diagnosis of MLP due to DLBCL. The patient received chemotherapy following surgery. So far, at 6 months of follow-up, Patient is doing well. CLINICAL DISCUSSION: Malignant tumors of the small intestine are unusual, with non-specific clinical presentation. Although ultrasound (US), CT, FDG-PET/CT and endoscopic evaluation are essential modalities for the diagnosis of intestinal polyposis. Final diagnosis of MLP can only be confirmed after histopathological examination and immunohistochemistry studies. Surgical resection followed by appropriate chemotherapy is the treatment of choice. CONCLUSIONS: MLP due to DLBCL has rarely been described in young patients under the age of 18 years. We should keep a high index of suspicion for malignant GI lymphoma in cases of intussusception, especially in older children.

Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation.

The heterogeneity of exosomal populations has hindered our understanding of their biogenesis, molecular composition, biodistribution and functions. By employing asymmetric flow field-flow fractionation (AF4), we identified two exosome subpopulations (large exosome vesicles, Exo-L, 90-120 nm; small exosome vesicles, Exo-S, 60-80 nm) and discovered an abundant population of non-membranous nanoparticles termed 'exomeres' (~35 nm). Exomere proteomic profiling revealed an enrichment in metabolic enzymes and hypoxia, microtubule and coagulation proteins as well as specific pathways, such as glycolysis and mTOR signalling. Exo-S and Exo-L contained proteins involved in endosomal function and secretion pathways, and mitotic spindle and IL-2/STAT5 signalling pathways, respectively. Exo-S, Exo-L and exomeres each had unique N-glycosylation, protein, lipid, DNA and RNA profiles and biophysical properties. These three nanoparticle subsets demonstrated diverse organ biodistribution patterns, suggesting distinct biological functions. This study demonstrates that AF4 can serve as an improved analytical tool for isolating extracellular vesicles and addressing the complexities of heterogeneous nanoparticle subpopulations.

Primary hydatid cyst of pancreas: Case report and review of literature.

INTRODUCTION: Hydatid disease is caused by the larval stage of Echinococcus granulosus. It most commonly affects the liver and lung. Pancreatic hydatid cyst (PHC) is very rare with incidence of 0.14%-2%. PRESENTATION OF CASE: A 40year old lady presented with epigastric pain for last 3 months. A 5×5cm abdominal lump occupying the epigastric and left hypochondrial region was noted on physical examination. Ultrasonography (USG) and Contrast enhanced Computed Tomogrpahy (CT) revealed a 55×57mm cystic structure in the pancreatic body. Endoscopic ultrasound guided fluid aspiration cytology revealed normal Carcinoembryonic antigen and Amylase levels. Cytological examination was noncontributory. During open surgical exploration, it was found to be a hydatid cyst. After irrigation with scolicidal agent and evacuation of cystic contents, Partial cystectomy with external drainage was done. Histopathological biopsy revealed Hydatid cyst. Post-operative ELISA (Enzyme linked immunosorbent assay) for Echinococcal antigen was positive. DISCUSSION: PHC is a rare entity. Most common mode of spread is hematogenous. Cysts in pancreatic head can present as obstructive jaundice. Cysts in body and tail are usually asymptomatic. USG, CT and Hydaitd serology can help in diagnosis and monitoring recurrence. Surgical exploration is treatment of choice. Options include pericystectomy, partial cystectomy+/- external drainage/omentopexy, marsupialization or cysto-enterostomy. Preoperative and Post-operative anti helminthic (Albendazole) is recommended. CONCLUSION: PHC can masquerade as pseudocyst or cystic neoplasm of pancreas. It should always be considered in the differential diagnosis of cystic pancreatic lesion in patients from endemic regions.

Laparoscopic cholecystectomy in a 2 year old male child with choleilithiasis and recurrent right hypochondrial pain: Case report and review of literature.

INTRODUCTION: Gallstone disease has been considered an uncommon entity in children and infants, but its incidence is reportedly increasing which may be attributed to widespread use of diagnostic imaging (ultrasonography). PRESENTATION OF CASE: An apparently healthy 2 year old male child presented to our Outpatient department with chief complaint of recurrent abdominal pain. The episodes of pain were acute in onset and associated with vomiting. As per the complete examination and findings, a diagnosis of chronic calculous cholecystitis was made. A four port laparoscopic cholecystectomy was done. DISCUSSION: The incidence of gallstones in children in India has not been sufficiently studied. The incidence of gallstone disease in India was found to be 0.3% with the incidence in age group 0-10 being less than 0.1%. In contrast to adult gallstone disease, it has been found that there is no female preponderance in gallstone diseases of infancy. Also, the majority of children having increased haemoglobin turnover develop pigment stones only after 5 years of age. CONCLUSION: The probability of gallstone disease in infants and young children should not be ignored. Gall stones should always be considered as a differential diagnosis when young patients present with complaints of abdominal pain.

Lysosomal proteolysis and autophagy require presenilin 1 and are disrupted by Alzheimer-related PS1 mutations.

Macroautophagy is a lysosomal degradative pathway essential for neuron survival. Here, we show that macroautophagy requires the Alzheimer's disease (AD)-related protein presenilin-1 (PS1). In PS1 null blastocysts, neurons from mice hypomorphic for PS1 or conditionally depleted of PS1, substrate proteolysis and autophagosome clearance during macroautophagy are prevented as a result of a selective impairment of autolysosome acidification and cathepsin activation. These deficits are caused by failed PS1-dependent targeting of the v-ATPase V0a1 subunit to lysosomes. N-glycosylation of the V0a1 subunit, essential for its efficient ER-to-lysosome delivery, requires the selective binding of PS1 holoprotein to the unglycosylated subunit and the Sec61alpha/oligosaccharyltransferase complex. PS1 mutations causing early-onset AD produce a similar lysosomal/autophagy phenotype in fibroblasts from AD patients. PS1 is therefore essential for v-ATPase targeting to lysosomes, lysosome acidification, and proteolysis during autophagy. Defective lysosomal proteolysis represents a basis for pathogenic protein accumulations and neuronal cell death in AD and suggests previously unidentified therapeutic targets.

IKK phosphorylates Huntingtin and targets it for degradation by the proteasome and lysosome.

Expansion of the polyglutamine repeat within the protein Huntingtin (Htt) causes Huntington's disease, a neurodegenerative disease associated with aging and the accumulation of mutant Htt in diseased neurons. Understanding the mechanisms that influence Htt cellular degradation may target treatments designed to activate mutant Htt clearance pathways. We find that Htt is phosphorylated by the inflammatory kinase IKK, enhancing its normal clearance by the proteasome and lysosome. Phosphorylation of Htt regulates additional post-translational modifications, including Htt ubiquitination, SUMOylation, and acetylation, and increases Htt nuclear localization, cleavage, and clearance mediated by lysosomal-associated membrane protein 2A and Hsc70. We propose that IKK activates mutant Htt clearance until an age-related loss of proteasome/lysosome function promotes accumulation of toxic post-translationally modified mutant Htt. Thus, IKK activation may modulate mutant Htt neurotoxicity depending on the cell's ability to degrade the modified species.

Constitutive activation of chaperone-mediated autophagy in cells with impaired macroautophagy.

Three different types of autophagy-macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA)-contribute to degradation of intracellular components in lysosomes in mammalian cells. Although some level of basal macroautophagy and CMA activities has been described in different cell types and tissues, these two pathways are maximally activated under stress conditions. Activation of these two pathways is often sequential, suggesting the existence of some level of cross-talk between both stress-related autophagic pathways. In this work, we analyze the consequences of blockage of macroautophagy on CMA activity. Using mouse embryonic fibroblasts deficient in Atg5, an autophagy-related protein required for autophagosome formation, we have found that blockage of macroautophagy leads to up-regulation of CMA, even under basal conditions. Interestingly, different mechanisms contribute to the observed changes in CMA-related proteins and the consequent activation of CMA during basal and stress conditions in these macroautophagy-deficient cells. This work supports a direct cross-talk between these two forms of autophagy, and it identifies changes in the lysosomal compartment that underlie the basis for the communication between both autophagic pathways.

Early cellular changes after blockage of chaperone-mediated autophagy.

Cytosolic proteins can be selectively degraded in lysosomes by chaperone-mediated autophagy (CMA), an autophagic pathway maximally activated under stress. In previous works we have demonstrated the existence of a cross-talk between CMA and macroautophagy, the other stress-related autophagic pathway responsible for the "in bulk" degradation of whole regions of the cytosol and for organelle turnover. We found that chronic blockage of CMA, as the one described in aging cells, results in constitutive activation of macroautophagy, supporting that one pathway may compensate for the other. In this work we have investigated the series of early cellular events that precede the activation of macroautophagy upon CMA blockage and the consequences of this blockage on cellular homeostasis. Shortly after CMA blockage, we have found functional alterations in macroautophagy and the ubiquitin-proteasome system, that are progressively corrected as CMA blockage persists. Basal macroautophagic activity remains initially unaltered, but we observed a delay in its activation in response to serum removal, a well characterized inducer for this pathway. Slower degradation of short-lived proteins, and a transient decrease in some of the proteasome proteolytic activities are also evident in the first stages of CMA blockage. This global alteration of the proteolytic systems supports the coordinated functioning of all of them, and seems responsible for the intracellular accumulation of altered proteins. Based on the time-course of the cellular changes, we propose that a minimal threshold of these toxic products needs to accumulate in order to constitutively activate macroautophagy and thus return cellular homeostasis to normal.

Dopamine-modified alpha-synuclein blocks chaperone-mediated autophagy.

Altered degradation of alpha-synuclein (alpha-syn) has been implicated in the pathogenesis of Parkinson disease (PD). We have shown that alpha-syn can be degraded via chaperone-mediated autophagy (CMA), a selective lysosomal mechanism for degradation of cytosolic proteins. Pathogenic mutants of alpha-syn block lysosomal translocation, impairing their own degradation along with that of other CMA substrates. While pathogenic alpha-syn mutations are rare, alpha-syn undergoes posttranslational modifications, which may underlie its accumulation in cytosolic aggregates in most forms of PD. Using mouse ventral medial neuron cultures, SH-SY5Y cells in culture, and isolated mouse lysosomes, we have found that most of these posttranslational modifications of alpha-syn impair degradation of this protein by CMA but do not affect degradation of other substrates. Dopamine-modified alpha-syn, however, is not only poorly degraded by CMA but also blocks degradation of other substrates by this pathway. As blockage of CMA increases cellular vulnerability to stressors, we propose that dopamine-induced autophagic inhibition could explain the selective degeneration of PD dopaminergic neurons.

Loss of macroautophagy promotes or prevents fibroblast apoptosis depending on the death stimulus.

Macroautophagy has been implicated as a mechanism of cell death. However, the relationship between this degradative pathway and cell death is unclear as macroautophagy has been shown recently to protect against apoptosis. To better define the interplay between these two critical cellular processes, we determined whether inhibition of macroautophagy could have both pro-apoptotic and anti-apoptotic effects in the same cell. Embryonic fibroblasts from mice with a knock-out of the essential macroautophagy gene atg5 were treated with activators of the extrinsic and intrinsic death pathways. Loss of macroautophagy sensitized these cells to caspase-dependent apoptosis from the death receptor ligands Fas and tumor necrosis factor-alpha (TNF-alpha). Atg5-/- mouse embryonic fibroblasts had increased activation of the mitochondrial death pathway in response to Fas/TNF-alpha in concert with decreased ATP levels. Fas/TNF-alpha treatment failed to up-regulate macroautophagy, and in fact, decreased activity at late time points. In contrast to their sensitization to Fas/TNF-alpha, Atg5-/- cells were resistant to death from menadione and UV light. In the absence of macroautophagy, an up-regulation of chaperone-mediated autophagy induced resistance to these stressors. These results demonstrate that inhibition of macroautophagy can promote or prevent apoptosis in the same cell and that the response is governed by the nature of the death stimulus and compensatory changes in other forms of autophagy. Experimental findings that an inhibition of macroautophagy blocks apoptosis do not prove that autophagy mediates cell death as this effect may result from the protective up-regulation of other autophagic pathways such as chaperone-mediated autophagy.

Altered dynamics of the lysosomal receptor for chaperone-mediated autophagy with age.

Rates of autophagy, the mechanism responsible for lysosomal clearance of cellular components, decrease with age. We have previously described an age-related decline in chaperone-mediated autophagy (CMA), a selective form of autophagy, by which particular cytosolic proteins are delivered to lysosomes after binding to the lysosome-associated membrane protein type 2A (LAMP-2A), a receptor for this pathway. Rates of CMA decrease with age because of a decrease in the levels of LAMP-2A. In this work we have investigated the reasons for the reduced levels of LAMP-2A with age. While transcriptional rates of LAMP-2A remain unchanged with age, the dynamics and stability of the receptor in the lysosomal compartment are altered. The mobilization of the lysosomal lumenal LAMP-2A to the membrane when CMA is activated is altered in lysosomes from old animals, leading to the presence of an unstable pool of lumenal LAMP-2A. By contrast, the regulated cleavage of LAMP-2A at the lysosomal membrane is reduced owing to altered association of the receptor and the protease responsible for its cleavage to particular membrane microdomain regions. We conclude that age-related changes at the lysosomal membrane are responsible for the altered turnover of the CMA receptor in old organisms and the consequent decline in this pathway.

Lysosome membrane lipid microdomains: novel regulators of chaperone-mediated autophagy.

Chaperone-mediated autophagy (CMA) is a selective mechanism for the degradation of soluble cytosolic proteins in lysosomes. The limiting step of this type of autophagy is the binding of substrates to the lysosome-associated membrane protein type 2A (LAMP-2A). In this work, we identify a dynamic subcompartmentalization of LAMP-2A in the lysosomal membrane, which underlies the molecular basis for the regulation of LAMP-2A function in CMA. A percentage of LAMP-2A localizes in discrete lysosomal membrane regions during resting conditions, but it exits these regions during CMA activation. Disruption of these regions by cholesterol-depleting agents or expression of a mutant LAMP-2A excluded from these regions enhances CMA activity, whereas loading of lysosomes with cholesterol significantly reduces CMA. Organization of LAMP-2A into multimeric complexes, required for translocation of substrates into lysosomes via CMA, only occurs outside the lipid-enriched membrane microdomains, whereas the LAMP-2A located within these regions is susceptible to proteolytic cleavage and degradation. Our results support that changes in the dynamic distribution of LAMP-2A into and out of discrete microdomains of the lysosomal membrane contribute to regulate CMA.

Lysosomal chat maintains the balance.

The original idea that each protein follows a particular proteolytic pathway for its degradation is no longer supported. Instead, different proteolytic systems can simultaneously contribute to the degradation of a particular protein, or they can alternate in this task depending, for the most part, on the cellular conditions. It is thus reasonable to expect that some level of communication exists among different proteolytic systems to orchestrate these coordinated activities. Direct cross-talk between two forms of autophagy, macroautophagy and chaperone-mediated autophagy (CMA) has been recently demonstrated. Cells respond to blockage of CMA by upregulating macroautophagy. Although macroautophagy cannot completely substitute for the lack of CMA, the partial redundancy between both pathways allows some level of compensation, enough to maintain protein degradation and preserve cell homeostasis. Understanding the cross-talk among different autophagic pathways and with other proteolytic systems is important to predict the type of compensatory mechanisms that could be elicited in response to failure of one of these systems, and to understand the consequences that manipulating one of these pathways for therapeutic purposes could have on the activity of the other pathways.

Autophagic defects in aging: looking for an "emergency exit"?

The ability of cells to renew their intracellular components and get rid of undesired or altered molecules decreases with age. Failure of autophagy is considered one of the main reasons for the build up of damaged components in the tissues of old organisms. We have recently shown that, declined activity of chaperone-mediated autophagy, a selective type of autophagy particularly impaired in aging, increases cell's vulnerability to stressors. This finding supports that, added to its role in cellular clean up, chaperone-mediated autophagy is an essential component of the cellular response to stress. Failure to perform this function with age could underlie the inability of old cells to adapt to stress conditions, and explain the accelerated course of some protein conformational disorders, such as Parkinson's disease, as affected individuals age.

Chaperone-mediated autophagy in aging and disease.

Different mechanisms target intracellular components for their degradation into lysosomes through what is known as autophagy. In mammals, three main forms of autophagy have been described: macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA). CMA is the only autophagic pathway that allows selective degradation of soluble proteins in lysosomes. In contrast to the other mammalian forms of autophagy, CMA does not require vesicle formation or major changes in the lysosomal membrane. Instead, substrate proteins directly cross the lysosomal membrane to reach the lumen, where they are rapidly degraded. The substrate proteins are targeted to the lysosomal membrane by recognition of a targeting motif (a KFERQ-like motif), by a chaperone complex, consisting of hsc70 and its cochaperones, in the cytoplasm. Once at the lysosomal membrane, the protein interacts with a lysosomal receptor for this pathway, lysosomal associated membrane protein type 2A (LAMP-2A), and it is translocated across the membrane into the lysosomal lumen assisted by a lysosome resident chaperone. These two characteristics--selectivity and direct substrate translocation--determine the particular role of CMA in different physiological and pathological conditions. In this chapter, we cover current findings on the molecular mechanisms for CMA and the possible pathophysiological relevance of this selective lysosomal degradation.

Autophagy is disrupted in a knock-in mouse model of juvenile neuronal ceroid lipofuscinosis.

Juvenile neuronal ceroid lipofuscinosis is caused by mutation of a novel, endosomal/lysosomal membrane protein encoded by CLN3. The observation that the mitochondrial ATPase subunit c protein accumulates in this disease suggests that autophagy, a pathway that regulates mitochondrial turnover, may be disrupted. To test this hypothesis, we examined the autophagic pathway in Cln3(Deltaex7/8) knock-in mice and CbCln3(Deltaex7/8) cerebellar cells, accurate genetic models of juvenile neuronal ceroid lipofuscinosis. In homozygous knock-in mice, we found that the autophagy marker LC3-II was increased, and mammalian target of rapamycin was down-regulated. Moreover, isolated autophagic vacuoles and lysosomes from homozygous knock-in mice were less mature in their ultrastructural morphology than the wild-type organelles, and subunit c accumulated in autophagic vacuoles. Intriguingly, we also observed subunit c accumulation in autophagic vacuoles in normal aging mice. Upon further investigation of the autophagic pathway in homozygous knock-in cerebellar cells, we found that LC3-positive vesicles were altered and overlap of endocytic and lysosomal dyes was reduced when autophagy was stimulated, compared with wildtype cells. Surprisingly, however, stimulation of autophagy did not significantly impact cell survival, but inhibition of autophagy led to cell death. Together these observations suggest that autophagy is disrupted in juvenile neuronal ceroid lipofuscinosis, likely at the level of autophagic vacuolar maturation, and that activation of autophagy may be a prosurvival feedback response in the disease process.

Consequences of the selective blockage of chaperone-mediated autophagy.

Chaperone-mediated autophagy (CMA) is a selective pathway for the degradation of cytosolic proteins in lysosomes. CMA declines with age because of a decrease in the levels of lysosome-associated membrane protein (LAMP) type 2A, a lysosomal receptor for this pathway. We have selectively blocked the expression of LAMP-2A in mouse fibroblasts in culture and analyzed the cellular consequences of reduced CMA activity. CMA-defective cells maintain normal rates of long-lived protein degradation by up-regulating macroautophagy, the major form of autophagy. Constitutive up-regulation of macroautophagy is unable, however, to compensate for all CMA functions. Thus, CMA-defective cells are more sensitive to stressors, suggesting that, although protein turnover is maintained, the selectivity of CMA is necessary as part of the cellular response to stress. Our results also denote the existence of cross-talk among different forms of autophagy.

Pathophysiology of chaperone-mediated autophagy.

In contrast to the classically described "in bulk" lysosomal degradation, the first evidence for selective degradation of cytosolic proteins in lysosomes was presented more than 20 years ago. Throughout this time, we have gained a better understanding about this process, now known as chaperone-mediated autophagy (CMA). The identification of new substrates for CMA and novel components, in both the cytosol and the lysosomes, along with better insights on how CMA is regulated, have all helped to shape the possible physiological roles of CMA. We review here different intracellular functions of CMA that arise from its unique characteristics when compared to other forms of autophagy. In view of these functions, we discuss the relevance of the changes in CMA activity in aging and in different pathological conditions.