Accumulation Of Autophagic Vesicles Research Articles

Raising awareness of TBC1 domain‐containing kinase (TBCK) epileptic encephalopathy among Puerto Rican children

AbstractBackgroundTBC1 domain‐containing kinase (TBCK) syndrome is a rare autosomal recessive genetic disorder that presents with infantile hypotonia, intellectual disability, motor impairment, and drug‐resistant epilepsy. The abnormal TBCK protein alters the mammalian target of rapamycin complex 1 (mTORC1), leading to accumulation of autophagic vesicles within fibroblasts. Thirty‐five previously reported individuals with TBCK were identified.MethodsWe present four children with TBCK due to the p.R126X (p.ARG126*) mutation, a variant predominating among children of Puerto Rican descent. This mutation has been associated with TBCK‐encephaloneuropathy or Boricua syndrome.DiscussionThese four of our patients were diagnosed within a span of 6 months, suggesting that the prevalence of the syndrome could be higher than expected among individuals from Puerto Rico. We believe this finding was largely due to the increasing availability and affordability of modern genetic testing in Puerto Rico, illustrating the importance of equitable access to genetic testing to all pediatric patients with similar symptomatology. Prompt diagnosis facilitates appropriate medical management and prognosis counseling.

Lysosomal dysfunction in Schwann cells is involved in bortezomib-induced peripheral neurotoxicity.

Bortezomib (BTZ) is a proteasome inhibitor serves as a first-line drug for multiple myeloma treatment. BTZ-induced peripheral neuropathy (BIPN) is the most common adverse effect of BTZ with an incidence as high as 40-60%. However, the pathological mechanisms underlying BIPN remain largely unclear. BTZ leads to dramatic Schwann cell demyelination in sciatic nerves. Previous studies implied that myelin debris was predominantly degraded via autophagy-lysosome pathway in Schwann cells. However, the association of autophagy with BIPN has not been made. Mice were treated with BTZ (2mg/kg, i.v.) on Day1 and Day4 each week for continuous 4weeks. BTZ-treated mice showed enhanced mechanical hyperalgesia, decreased tail nerve conduction and sciatic nerve demyelination. Unexpectedly, BTZ led to the accumulation of autophagic vesicles, LC3-II and p62 in the sciatic nerve. Moreover, BTZ blocked autophagic flux in RSC96 Schwann cells as determined by mcherry-GFP-LC3 assay, suggesting BTZ may impair lysosomal function rather than inducing autophagy in Schwann cells. BTZ significantly reduced the lysosomal activity in Schwann cells as determined by reduced LysoTracker Red and DQ-Red-BSA staining and increased the level of immature Cathepsin B (CTSB). Remarkably, lysosomal activators PP242 and Torin1, significantly reversed the blockage of autophagic flux by BTZ. We further verified that Torin1 rescued the demyelination, nerve conduction and reduced the mechanical hyperalgesia in BIPN mice. Additionally, Torin1 did not compromise the efficacy of BTZ in suppressing multiple myeloma RPMI8226 cell. Taken together, we identified that lysosomal dysfunction in Schwann cells caused by BTZ is involved in the BIPN pathology. Improved lysosomal function in Schwann cells can be a promising strategy for BIPN treatment.

Drosophila as a model to study autophagy in neurodegenerative diseases and digestive tract.

Autophagy regulates cellular homeostasis by degrading and recycling cytosolic components and damaged organelles. Disruption of autophagic flux has been shown to induce or facilitate neurodegeneration and accumulation of autophagic vesicles is overt in neurodegenerative diseases. The fruit fly Drosophila has been used as a model system to identify new factors that regulate physiology and disease. Here we provide a historical perspective of how the fly models have offered mechanistic evidence to understand the role of autophagy in neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, Charcot-Marie-Tooth neuropathy, and polyglutamine disorders. Autophagy also plays a pivotal role in maintaining tissue homeostasis and protecting organism health. The gastrointestinal tract regulates organism health by modulating food intake, energy balance, and immunity. Growing evidence is strengthening the link between autophagy and digestive tract health in recent years. Here, we also discuss how the fly models have advanced the understanding of digestive physiology regulated by autophagy.

Chloroplast proteotoxic stress-induced autophagy is involved in the degradation of chloroplast proteins in Chlamydomonas reinhardtii.

Intraorganellar proteases and cytoplasmic proteolytic systems such as autophagy orchestrate the degradation of organellar proteins to ensure organelle homeostasis in eukaryotic cells. The green alga Chlamydomonas reinhardtii is an ideal unicellular model organism for elucidating the mechanisms maintaining proteostasis in chloroplasts. However, the autophagic pathways targeting the photosynthetic organelles of these algae have not been clearly elucidated. Here, we explored the role of autophagy in chloroplast protein degradation in Chlamydomonas cells. We labeled the chloroplast protein Rubisco small subunit (RBCS) with the yellow fluorescent protein Venus in a Chlamydomonas strain in which expression of the chloroplast gene clpP1, encoding a major catalytic subunit of the chloroplast Clp protease, can be conditionally repressed to selectively perturb chloroplast protein homeostasis. We observed transport of both nucleus-encoded RBCS-Venus fusion protein and chloroplast-encoded Rubisco large subunit (rbcL) from the chloroplast to the vacuoles in response to chloroplast proteotoxic stress induced by clpP1 inhibition. This process was retarded by the addition of autophagy inhibitors. Biochemical detection of lytic cleavage of RBCS-Venus supported the notion that Rubisco is degraded in the vacuoles via autophagy. Electron microscopy revealed vacuolar accumulation of autophagic vesicles and exposed their ultrastructure during repression of clpP1 expression. Treatment with an autophagy activator also induced chloroplast autophagy. These results indicate that autophagy contributes to chloroplast protein degradation in Chlamydomonas cells.

Retraction: Chloroplast proteotoxic stress-induced autophagy is involved in the degradation of chloroplast proteins in Chlamydomonas reinhardtii.

Intraorganellar proteases and cytoplasmic proteolytic systems such as autophagy orchestrate the degradation of organellar proteins to ensure organelle homeostasis in eukaryotic cells. The green alga Chlamydomonas reinhardtii is an ideal unicellular model organism for elucidating the mechanisms maintaining proteostasis in chloroplasts. However, the autophagic pathways targeting the photosynthetic organelles of these algae have not been clearly elucidated. Here, we explored the role of autophagy in chloroplast protein degradation in Chlamydomonas cells. We labeled the chloroplast protein Rubisco small subunit (RBCS) with the yellow fluorescent protein Venus in a Chlamydomonas strain in which expression of the chloroplast gene clpP1, encoding a major catalytic subunit of the chloroplast Clp protease, can be conditionally repressed to selectively perturb chloroplast protein homeostasis. We observed transport of both nucleus-encoded RBCS-Venus fusion protein and chloroplast-encoded Rubisco large subunit (rbcL) from the chloroplast to the vacuoles in response to chloroplast proteotoxic stress induced by clpP1 inhibition. This process was retarded by the addition of autophagy inhibitors. Biochemical detection of lytic cleavage of RBCS-Venus supported the notion that Rubisco is degraded in the vacuoles via autophagy. Electron microscopy revealed vacuolar accumulation of autophagic vesicles and exposed their ultrastructure during repression of clpP1 expression. Treatment with an autophagy activator also induced chloroplast autophagy. These results indicate that autophagy contributes to chloroplast protein degradation in Chlamydomonas cells.

Protein Homeostasis Networks and the Use of Yeast to Guide Interventions in Alzheimer's Disease.

Alzheimer’s Disease (AD) is a progressive multifactorial age-related neurodegenerative disorder that causes the majority of deaths due to dementia in the elderly. Although various risk factors have been found to be associated with AD progression, the cause of the disease is still unresolved. The loss of proteostasis is one of the major causes of AD: it is evident by aggregation of misfolded proteins, lipid homeostasis disruption, accumulation of autophagic vesicles, and oxidative damage during the disease progression. Different models have been developed to study AD, one of which is a yeast model. Yeasts are simple unicellular eukaryotic cells that have provided great insights into human cell biology. Various yeast models, including unmodified and genetically modified yeasts, have been established for studying AD and have provided significant amount of information on AD pathology and potential interventions. The conservation of various human biological processes, including signal transduction, energy metabolism, protein homeostasis, stress responses, oxidative phosphorylation, vesicle trafficking, apoptosis, endocytosis, and ageing, renders yeast a fascinating, powerful model for AD. In addition, the easy manipulation of the yeast genome and availability of methods to evaluate yeast cells rapidly in high throughput technological platforms strengthen the rationale of using yeast as a model. This review focuses on the description of the proteostasis network in yeast and its comparison with the human proteostasis network. It further elaborates on the AD-associated proteostasis failure and applications of the yeast proteostasis network to understand AD pathology and its potential to guide interventions against AD.

Abstract B20: STK3/4-mediated phosphorylation of LC3B regulates directional intracellular transport of autophagic vesicles

Abstract Autophagy is a fundamental catabolic process that allows the degradation of cytosolic components to supply the cell with nutrients in response to starvation, among other functions. Recently, our lab discovered that the Hippo pathway members, STK3 and STK4, phosphorylate the autophagy protein LC3B, a key component of autophagic vesicles that participates in specifying their content and their intracellular transport. Depletion of STK3/4 kinases or generation of an LC3B phospho-null mutant (T50A) causes a block of the autophagy process evidenced by an accumulation of autophagic vesicles throughout the cell. However, these studies did not clarify which step of the autophagy process was impaired. To address this question, we employed proteomics to analyze LC3B-binding partners affected by LC3B phosphorylation, and identified multiple proteins implicated in intracellular vesicle transport. Accordingly, we hypothesized that LC3B phosphorylation may affect transport of autophagic vesicles. We tested this hypothesis directly by live-cell imaging in multiple cell types and found that the absence of LC3B phosphorylation was accompanied by an aberrantly high transport of LC3B-positive autophagic vesicles toward the periphery of the cell, and a low transport of the vesicles toward the perinuclear area, where the autophagy process is normally completed. Moreover, starvation-induced perinuclear positioning of autophagic vesicles was prevented in the absence of LC3B phosphorylation. Collectively, these findings indicate that STK3/4-mediated phosphorylation of LC3B regulates the directional transport of autophagic vesicles and this may promote autophagy completion. Notably, this regulatory axis may be triggered when the nutrient availability is low and autophagy is induced. Understanding how input signals regulate STK3/4, and in turn control autophagy, may prove important to develop therapies against diseases such as cancer. Citation Format: Jose L. Nieto-Torres, Sean-Luc Shanahan, Romain Chassefeyre, Sara Landeras-Bueno, Lee R. Lee, Sviatlana Zaretski, Sandra Encalada, Malene Hansen. STK3/4-mediated phosphorylation of LC3B regulates directional intracellular transport of autophagic vesicles [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr B20.

LRRK2 inhibition prevents endolysosomal deficits seen in human Parkinson's disease

LRRK2 has been implicated in endolysosomal function and likely plays a central role in idiopathic Parkinson's disease (iPD). In iPD, dopaminergic neurons within the substantia nigra are characterized by increased LRRK2 kinase activity, endolysosomal deficits, and accumulation of autophagic vesicles with incompletely degraded substrates, including α-synuclein. Although LRRK2 has been implicated in endolysosomal and autophagic function, it remains unclear whether inhibition of LRRK2 kinase activity can prevent endolysosomal deficits or reduce dopaminergic neurodegeneration. In this study, we characterized the endolysosomal and autophagic defects in surviving dopaminergic neurons of iPD patient brain tissue. We next showed that these defects could be reproduced reliably in vivo using the rotenone model of iPD. Results suggested that there was impaired endosomal maturation, resulting in lysosomal dysfunction and deficits in protein degradation. A highly selective, brain-penetrant LRRK2 kinase inhibitor not only improved apparent endosomal maturation and lysosomal function, but also prevented rotenone-induced neurodegeneration in vivo. The fact that a LRRK2 kinase inhibitor was capable of preventing the neuropathological and endolysosomal abnormalities observed in human iPD suggests that LRRK2 inhibitors may have broad therapeutic utility in iPD, not only in those who carry a LRRK2 mutation.

Sec20 is Required for Autophagic and Endocytic Degradation Independent of Golgi-ER Retrograde Transport.

Endocytosis and autophagy are evolutionarily conserved degradative processes in all eukaryotes. Both pathways converge to the lysosome where cargo is degraded. Improper lysosomal degradation is observed in many human pathologies, so its regulatory mechanisms are important to understand. Sec20/BNIP1 (BCL2/adenovirus E1B 19 kDa protein-interacting protein 1) is a BH3 (Bcl-2 homology 3) domain-containing SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptors) protein that has been suggested to promote Golgi-ER retrograde transport, mitochondrial fission, apoptosis and mitophagy in yeast and vertebrates. Here, we show that loss of Sec20 in Drosophila fat cells causes the accumulation of autophagic vesicles and prevents proper lysosomal acidification and degradation during bulk, starvation-induced autophagy. Furthermore, Sec20 knockdown leads to the enlargement of late endosomes and accumulation of defective endolysosomes in larval Drosophila nephrocytes. Importantly, the loss of Syx18 (Syntaxin 18), one of the known partners of Sec20, led to similar changes in nephrocytes and fat cells. Interestingly. Sec20 appears to function independent of its role in Golgi-ER retrograde transport in regulating lysosomal degradation, as the loss of its other partner SNAREs Use1 (Unconventional SNARE In The ER 1) and Sec22 or tethering factor Zw10 (Zeste white 10), which function together in the Golgi-ER pathway, does not cause defects in autophagy or endocytosis. Thus, our data identify a potential new transport route specific to lysosome biogenesis and function.

Abstract 43: Regulation of autophagy in bone metastatic breast cancer cells

Abstract Bone metastasis of breast cancer is a significant cause of patient mortality. Recent studies suggest that metastatic cancer cells induce autophagy to survive metabolic stress. During autophagy, cytoplasmic components and damaged organelles are captured by autophagosomes followed by lysosomal fusion and degradation, releasing metabolites as energy sources to meet metabolic demands. Although the components of the autophagy pathway have been well characterized, the regulatory mechanisms of autophagy in metastatic cancer cells remain unknown. Previously, we have shown that Runt-related transcription factor-2 (Runx2) promotes cell survival, bone metastasis, and osteolysis. Using a bone metastatic isogenic variant of breast cancer MDA-MB-231, we examined levels of the autophagosome specific marker LC3B to define the regulation of autophagy during bone metastasis. Additionally, we examined whether Runx2 regulates autophagy for increased cell survival in the bone microenvironment. Microscopic and biochemical studies showed elevated levels of autophagic flux among bone derived cells relative to parental breast cancer cells. Interestingly, we also observed that Runx2 enhanced the turnover of autophagic vesicles while Runx2 silencing resulted in accumulation of vesicles due to reduced turnover. Interestingly, Runx2 knockdown or inhibition of autophagy in bone derived cells increased AMPK levels suggesting higher levels of cellular stress as a consequence of impaired autophagy. In addition to AMPK activity, MAP kinase mutations have been demonstrated to result in constitutive activation of the autophagy pathway. Treatment with the MEK inhibitor PD184161 resulted in accumulation of LC3B-II in control cells. Furthermore, Runx2 knockdown in bone derived cells display lower levels of ERK activity relative to controls. These results suggest that Runx2/ERK signaling is critical for autophagy in metastatic breast cancer cells. Our mechanistic studies revealed that Runx2 promotes autophagy by increasing acetylation of α-tubulin sub-units of microtubules and enhancing trafficking of autophagic vesicles. Introduction of a mutant α-tubulin construct incapable of being acetylated resulted in the accumulation of autophagic vesicles in control cells, similar to silencing of Runx2. Inhibition of autophagy resulted in decreased adhesion, migration, and survival of Runx2 knockdown cells. Furthermore, analysis of LC3B protein in clinical breast cancer specimens and tumor xenografts revealed significant association between high Runx2 and low LC3B protein levels. Our studies reveal a novel regulatory mechanism of autophagy via Runx2 and provide insights into the role of autophagy in bone metastatic breast cancer cells. Citation Format: Ahmad H. Othman, Manish Tandon, Vivek Ashok, Marcus Winogradzki, Gary Stein, Jitesh Pratap. Regulation of autophagy in bone metastatic breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 43.

Autophagic dysfunction in patients with Papillon-Lefèvre syndrome is restored by recombinant cathepsin C treatment

Cathepsin C (CatC) is a lysosomal enzyme involved in activation of serine proteases from immune and inflammatory cells. Several loss-of-function mutations in the CatC gene have been shown to be the genetic mark of Papillon-Lefèvre syndrome (PLS), a rare autosomal recessive disease characterized by severe early-onset periodontitis, palmoplantar hyperkeratosis, and increased susceptibility to infections. Deficiencies or dysfunction in other cathepsin family proteins, such as cathepsin B or D, have been associated with autophagic and lysosomal disorders. Here we characterized the basis for autophagic dysfunction in patients with PLS by analyzing skin fibroblasts derived from patients with several mutations in the CatC gene and reduced enzymatic activity. Skin fibroblasts were isolated from patients with PLS assessed by using genetic analysis. Authophagic flux dysfunction was evaluated by examining accumulation of p62/SQSTM1 and a bafilomycin assay. Ultrastructural analysis further confirmed abnormal accumulation of autophagic vesicles in mutant cells. Arecombinant CatC protein was produced by a baculovirus system in insect cell cultures. Mutant fibroblasts from patients with PLS showed alterations in oxidative/antioxidative status, reduced oxygen consumption, and a marked autophagic dysfunction associated with autophagosome accumulation. These alterations were accompanied by lysosomal permeabilization, cathepsin Brelease, and NLR family pyrin domain containing 3 (NLRP3) inflammasome activation. Treatment of mutant fibroblasts with recombinant CatC improved cell growth and autophagic flux and partially restored lysosomal permeabilization. Our data provide a novel molecular mechanism underlying PLS. Impaired autophagy caused by insufficient lysosomal function might represent a new therapeutic target for PLS.

Structural diversity of Burkholderia pseudomallei lipopolysaccharides affects innate immune signaling.

Burkholderia pseudomallei (Bp) causes the disease melioidosis. The main cause of mortality in this disease is septic shock triggered by the host responding to lipopolysaccharide (LPS) components of the Gram-negative outer membrane. Bp LPS is thought to be a weak inducer of the host immune system. LPS from several strains of Bp were purified and their ability to induce the inflammatory mediators TNF-α and iNOS in murine macrophages at low concentrations was investigated. Innate and adaptive immunity qPCR arrays were used to profile expression patterns of 84 gene targets in response to the different LPS types. Additional qPCR validation confirmed large differences in macrophage response. LPS from a high-virulence serotype B strain 576a and a virulent rough central nervous system tropic strain MSHR435 greatly induced the innate immune response indicating that the immunopathogenesis of these strains is different than in infections with strains similar to the prototype strain 1026b. The accumulation of autophagic vesicles was also increased in macrophages challenged with highly immunogenic Bp LPS. Gene induction and concomitant cytokine secretion profiles of human PBMCs in response to the various LPS were also investigated. MALDI-TOF/TOF was used to probe the lipid A portions of the LPS, indicating substantial structural differences that likely play a role in host response to LPS. These findings add to the evolving knowledge of host-response to bacterial LPS, which can be used to better understand septic shock in melioidosis patients and in the rational design of vaccines.

Abnormal cell-clearance and accumulation of autophagic vesicles in lymphocytes from patients affected with Ataxia-Teleangiectasia

Ataxia-Teleangiectasia (A-T) is a neurodegenerative disorder due to mutations in ATM gene. ATM in the nucleus ensures DNA repair, while its role in the cytosol is still poorly clarified. Abnormal autophagy has been documented in other neurodegenerative disorders, thus we evaluated whether alteration in this process may be involved in the pathogenesis of A-T by analyzing the autophagic vesicles and the genes implicated in the different stages of autophagy. Through transmission electron microscopy (TEM) and immunofluorescence analysis we observed an accumulation of APs associated with a LC3 puncta pattern, and a reduced number of ALs. We also documented an increased expression of genes involved in AP and lysosome biogenesis and function, and a decrease of Vps18 expression, involved in their vesicular trafficking and fusion. mTORC1-controlled proteins were hyperphosphorylated in A-T, in keeping with an increased mTOR inhibitory influence of autophagy. Betamethasone is able to promote the degradation of SQSTM1, a biomarker of autophagy. Collectively, our results indicate that in cells from A-T patients, the APs maturation is active, while the fusion between APs and lysosomes is inappropriate, thus implying abnormalities in the cell-clearance process. We also documented a positive effect of Betamethasone on molecules implicated in autophagosome degradation.

Autophagic and lysosomal defects in human tauopathies: analysis of post-mortem brain from patients with familial Alzheimer disease, corticobasal degeneration and progressive supranuclear palsy.

IntroductionThe accumulation of insoluble proteins within neurons and glia cells is a pathological hallmark of several neurodegenerative diseases. Abnormal aggregation of the microtubule-associated protein tau characterizes the neuropathology of tauopathies, such as Alzheimer disease (AD), corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP). An impairment of the lysosomal degradation pathway called macroautophagy, hereafter referred to as autophagy, could contribute to the accumulation of aggregated proteins. The role of autophagy in neurodegeneration has been intensively studied in the context of AD but there are few studies in other tauopathies and it is not known if defects in autophagy is a general feature of tauopathies. In the present study, we analysed autophagic and lysosomal markers in human post-mortem brain samples from patients with early-onset familial AD (FAD) with the APP Swedish mutation (APPswe), CBD and PSP and control individuals.ResultsFAD, CBD and PSP patients displayed an increase in LC3-positive vesicles in frontal cortex, indicating an accumulation of autophagic vesicles. Moreover, using double-immunohistochemistry and in situ proximity ligation assay, we observed colocalization of hyperphosphorylated tau with the autophagy marker LC3 in FAD, CBD and PSP patients but not in control individuals. Increased levels of the lysosomal marker LAMP1 was detected in FAD and CBD, and in addition Cathepsin D was diffusely spread in the cytoplasm in all tauopathies suggesting an impaired lysosomal integrity.ConclusionTaken together, our results indicate an accumulation of autophagic and lysosomal markers in human brain tissue from patients with primary tauopathies (CBD and PSP) as well as FAD, suggesting a defect of the autophagosome-lysosome pathway that may contribute to the development of tau pathology.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-016-0292-9) contains supplementary material, which is available to authorized users.

C-Jun N-terminal kinase activation by nitrobenzoxadiazoles leads to late-stage autophagy inhibition.

Background Nitrobenzoxadiazole derivatives (NBDs), including NBDHEX and the recently developed MC3181, are promising anticancer agents able to target glutathione transferase and inhibit both its catalytic activity and ability to sequester TNF-receptor associated factor 2 (TRAF2) and c-Jun N-terminal kinase (JNK). NBDs have been shown to impair the growth and survival of a broad-spectrum of tumor types, in vitro and in vivo. Herein, we evaluated the effects of the new compound MC3181 on U-2OS osteosarcoma cells and investigated the impact of both NBDHEX and MC3181 on autophagy.MethodsCell viability was evaluated by sulforhodamine B assay. The dissociation of the TRAF2-GSTP1-1 complex was detected by proximity ligation assay, while the phospho-activation of JNK was assessed by western blotting. The effects of NBDs on autophagy were evaluated by GFP-LC3 puncta formation, western blotting for LC3-II and p62, and LC3 turnover assay in the presence of bafilomycin A1. The role of JNK in the reduction of autophagic flux caused by NBDs was investigated using JNK1 shRNA-transfected cells. Fluorogenic caspase activity assay and flow cytometric analysis of DNA content were used to determine the cytotoxic effects of NBDs on JNK1-silenced cells.ResultsSimilar to NBDHEX, MC3181 reduced viability and activated TRAF2/JNK signaling in U-2OS cells. Moreover, NBDs induced the accumulation of autophagic vesicles and LC3-II while reducing both basal and nutritional stress-induced autophagic flux. Furthermore, increased levels of both LC3-II and the autophagy selective substrate p62 were observed in different tumor cell lines treated with NBDs, the concurrent increase of these markers being consistent with an impairment of autophagosome clearance. Autophagy inhibition by NBDs required JNK activity: NBDs caused autophagy inhibition and caspase-3 activation in JNK-positive U-2OS, but no autophagic flux inhibition or caspase-3 activation in JNK-silenced cells.ConclusionsOur demonstration that NBDs can act as late-phase autophagy inhibitors opens new opportunities to fully exploit their therapeutic potential. This may not rely solely on their effectiveness in inducing cell cycle arrest and apoptosis, but also on their ability to weaken the capacity of tumor cells to endure stress conditions via autophagy. In addition, this study provides evidence that JNK can participate in impairing autophagy.

Lysosomal reacidification via degradation of PLGA nanoparticles in a lipotoxic cardiomyopathy model

Event Abstract Back to Event Lysosomal reacidification via degradation of PLGA nanoparticles in a lipotoxic cardiomyopathy model Frederick M. Zasadny1, 2, James A. Ankrum1, 2 and E Dale Abel2, 3, 4 1 University of Iowa, Biomedical Engineering, United States 2 University of Iowa, Fraternal Order of Eagles Diabetes Research Center, United States 3 University of Iowa Carver College of Medicine, Internal Medicine, United States 4 University of Iowa, Biochemistry, United States Introduction: Lipotoxic cardiomyopathy is one of several cardiovascular complications that cause one third of deaths worldwide[1]. Specifically, obesity related lipid-overload impairment of autophagy causes pathophysiological changes in heart function and structure. There is an increase in fatty acid uptake and utilization in the heart that increases toxic lipid species[2]. These cardiotoxic lipid species have recently been shown to deacidify lysosomes in the heart, the terminus of autophagy[3]. When deacidified, the proteolytic enzymes in the lysosome cannot process autophagic vesicles. Thus, there is an accumulation of autophagic vesicles in heart muscle tissue which impairs cardiac function. Here, a therapeutic strategy utilizing poly(lactic-co-glycolic acid) (PLGA) nanoparticle degradation to reacidify the lysosome and revert the effects of cardiotoxic lipid species restoring autophagic flux in H9c2 cardiomyocytes is presented. Materials and Methods: Nanoparticle (NP) formulation consists of single emulsion solvent extraction evaporation. Namely, PLGA is dissolved in an organic solvent dichloromethane (DCM) (not miscible in water) which is then added to polyvinyl alcohol (PVA) (miscible in water). Upon emulsification of the solution, nanoparticles of PLGA surrounded by DCM are formed suspended in the PVA solution. DCM then evaporates leaving behind hardened nanoparticles in PVA. H9c2 cardiomyocytes (CM’s) were grown to 80% confluence in six well plates and then differentiated for four days with 10nM all-trans retinoic acid. On the third day of differentiation, CM’s were treated with PLGA NP’s for ~15 hours, upon which the cells were treated with 500uM palmitate (Palm) for 4 hours. After treatments, Western Blot, fluorescence microscopy and other assays assessed autophagic flux and PLGA NP activity. For microscopy, PLGA NP’s were labeled with DiD stain to enable colocalization studies with the lysosome (LysoTracker) and autophagic vesicle stains (Cyto-ID). Proteolytic enzyme (Cathepsin L) activity in the lysosome was assessed with Magic Red fluorescence, decreasing with palmitate treatment. Results and Discussion: H9c2 cardiomyocytes treated with palmitate increased Cyto-ID florescence of autophagic vesicles indicating correct modeling of obesity related lipid-overload impairment of autophagy, i.e., a blockade of autophagic flux. Colocalization studies with DiD and LysoTracker stains indicate that the PLGA NP’s are indeed phagocytosed and transported to the lysosome. PLGA NP treatment increased Cathepsin L activity when CM’s are also treated with Palm compared to just Palm treatment, as shown in Figure 1. This indicates that the hydrolytic degradation of PLGA NP’s into lactic and glycolic acid is functionally reacidifying the lysosomes in H9c2 cardiomyocytes and seemingly restoring Cathepsin L activity in the lysosome. Western blot indicates a decrease in the accumulation of autophagic vesicle protein build-up caused by palmitate treatment when co-treated with PLGA NP’s suggesting a restoration of autophagic flux. Conclusions: PLGA NP’s functionally revert obesity related lipid-overload impairment of autophagy in cardiomyocytes with respect to reacidification of impaired lysosomes indicated by restored and increased Cathepsin L activity. Future studies include optimization of PLGA NP design including the conjugation of cardiomyocyte specific moieties that will enable in vivo targeting of heart tissue.

Glutathione Depletion Induces Spermatogonial Cell Autophagy.

The development and survival of male germ cells depend on the antioxidant capacity of the seminiferous tubule. Glutathione (GSH) plays an important role in the antioxidant defenses of the spermatogenic epithelium. Autophagy can act as a pro-survival response during oxidative stress or nutrient deficiency. In this work, we evaluated whether autophagy is involved in spermatogonia-type germ cell survival during severe GSH deficiency. We showed that the disruption of GSH metabolism with l-buthionine-(S,R)-sulfoximine (BSO) decreased reduced (GSH), oxidized (GSSG) glutathione content, and GSH/GSSG ratio in germ cells, without altering reactive oxygen species production and cell viability, evaluated by 2',7'-dichlorodihydrofluorescein (DCF) fluorescence and exclusion of propidium iodide assays, respectively. Autophagy was assessed by processing the endogenous protein LC3I and observing its sub-cellular distribution. Immunoblot and immunofluorescence analysis showed a consistent increase in LC3II and accumulation of autophagic vesicles under GSH-depletion conditions. This condition did not show changes in the level of phosphorylation of AMP-activated protein kinase (AMPK) or the ATP content. A loss in S-glutathionylated protein pattern was also observed. However, inhibition of autophagy resulted in decreased ATP content and increased caspase-3/7 activity in GSH-depleted germ cells. These findings suggest that GSH deficiency triggers an AMPK-independent induction of autophagy in germ cells as an adaptive stress response.

A novel antitumour strategy using bidirectional autophagic vesicles accumulation via initiative induction and the terminal restraint of autophagic flux

Autophagy is a lysosomal degradation pathway that protects cancer cells from multiple endogenous and extraneous stresses, particularly during the pathogenesis of cancer. An autophagic balance exists in the tumour microenvironment. Appropriate disturbance to this balance may have therapeutic potential. Here, we report a novel antitumour strategy based on an autophagic catastrophic vacuolisation effect in tumour cells. We achieved this effect via initiative induction and the terminal restraint of autophagic flux. The TAT–Beclin 1 peptide (T–B) was constructed for the initiative induction of autophagic flux, whereas hydroxychloroquine (HCQ)-loaded liposomes (HCQ-Lip) were constructed for terminal restraint. We demonstrate that T–B, a new CPP tandem autophagy inducing peptide, effectively activates the autophagy signal at the early stage of the autophagy pathway. HCQ deacidified the lysosome during the final stage of autophagy. We combined T–B and HCQ-Lip to induce autophagic catastrophic vacuolisation and death in several tumour cell lines based on the idea of “broadening sources of income and reducing expenditure”. The co-treated group exhibited at least a 1.86-fold greater and up to 5.66-fold greater cytotoxic effect in vitro. In addition, this strategy showed at least a 2.0-fold tumour inhibitory effect compared to the other groups in vivo. Therefore, this bidirectional accumulation of autophagic vesicles exhibited potential efficacy for tumour treatment.

Longitudinal follow-up of autophagy and inflammation in brain of APPswePS1dE9 transgenic mice.

BackgroundIn recent years, studies have sought to understand the mechanisms involved in the alteration of autophagic flux in Alzheimer's disease (AD). Alongside the recent description of the impairment of lysosomal acidification, we wanted to study the relationships between inflammation and autophagy, two physiological components deregulated in AD. Therefore, a longitudinal study was performed in APPswePS1dE9 transgenic mice at three, six and twelve months of age.MethodsAutophagic markers (Beclin-1, p62 and LC3) and the activation of mammalian Target of Rapamycin (mTOR) signaling pathway were quantified by western blot. Cytokine levels (IL-1β, TNF-α and IL-6) were measured by ELISA. Transmission electron microscopy was performed to detect autophagic vacuoles. Mann-Whitney tests were used to compare wild-type (WT) versus APPswePS1dE9 mice. Longitudinal changes in parameters were analyzed with a Kruskal-Wallis test followed by a post-hoc Dunn’s test. Correlation between two parameters was assessed using a Spearman test.ResultsCompared to 12-month old WT mice, 12-month old APPswePS1dE9 mice had higher levels of IL-1β and TNF-α, a greater inhibition of the mTOR signaling pathway and lower levels of Beclin-1 expression both in cortex and hippocampus. Regarding the relationship of the various parameters in 12-month old APPswePS1dE9 mice, Beclin-1 rates were positively correlated with IL-1β and TNF-α levels. And, on the contrary, TNF-α levels were inversely correlated with the levels of mTOR activation. Altogether, these results suggest that inflammation could induce autophagy in APPswePS1dE9 mice. However, these transgenic mice displayed a large accumulation of autophagic vesicles within dystrophic neurons in cortex and hippocampus, indicating a terminal failure in the autophagic process.ConclusionsThis first demonstration of relationships between inflammation and autophagy in in vivo models of AD should be taken into account in new therapeutic strategies to prevent inflammation and/or stimulate autophagy in advanced neurodegenerative process such as AD.

Phospholipase D1 regulates autophagic flux and clearance of α-synuclein aggregates

Many neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, are characterized by abnormal accumulations of aggregated proteins. Brains in these diseases also show accumulation of autophagic vesicles in the neuronal cytoplasm, suggesting impairment of the autophagic process. As autophagy involves de novo membrane production and vesicle fusion, extensive changes in lipid molecules are necessary. However, the involvement of signaling lipid-modifying enzymes in autophagy and their roles in neurodegenerative diseases are not clear. Using specific inhibitor, we show that loss of phospholipase D1 (PLD1) activity resulted in an accumulation of microtubule-associated protein light chain 3 (LC3), p62, and polyubiquitinated proteins, signs representing malfunction in autophagic flux. Fluorescence and electron microscopic analyses demonstrated impaired fusion of autophagosomes with lysosomes, resulting in accumulation of autophagosomes. Within the cells with impaired autophagic flux, α-synuclein aggregates accumulated in autophagosomes. Knockdown of PLD1 expression using small interfering RNA also resulted in impaired autophagic flux and accumulation of α-synuclein aggregates in autophagosomes. Neuronal toxicity caused by α-synuclein accumulation was rescued by overexpression of PLD1; however, expression of activity-deficient mutant, PLD1-KRM, showed reduced rescue effects. Finally, we demonstrated that both PLD activity and expression levels were reduced in brain tissues of dementia with Lewy bodies (DLB) patients, whereas the amounts of α-synuclein and p62 were increased in the same tissue samples. Collectively, these results suggest that insufficient PLD activity, and therefore, the changes in phospholipid compositions within membranes, might be an important contributor to impaired autophagic process and protein accumulation in Lewy body diseases.