Showing papers in "Autophagy in 2014"

A phase I/II trial of hydroxychloroquine in conjunction with radiation therapy and concurrent and adjuvant temozolomide in patients with newly diagnosed glioblastoma multiforme

Abstract: Preclinical studies indicate autophagy inhibition with hydroxychloroquine (HCQ) can augment the efficacy of DNA-damaging therapy. The primary objective of this trial was to determine the maximum tolerated dose (MTD) and efficacy of HCQ in combination with radiation therapy (RT) and temozolomide (TMZ) for newly diagnosed glioblastoma (GB). A 3 + 3 phase I trial design followed by a noncomparative phase II study was conducted in GB patients after initial resection. Patients received HCQ (200 to 800 mg oral daily) with RT and concurrent and adjuvant TMZ. Quantitative electron microscopy and immunoblotting were used to assess changes in autophagic vacuoles (AVs) in peripheral blood mononuclear cells (PBMC). Population pharmacokinetic (PK) modeling enabled PK-pharmacodynamic correlations. Sixteen phase I subjects were evaluable for dose-limiting toxicities. At 800 mg HCQ/d, 3/3 subjects experienced Grade 3 and 4 neutropenia and thrombocytopenia, 1 with sepsis. HCQ 600 mg/d was found to be the MTD in this combination. The phase II cohort (n = 76) had a median survival of 15.6 mos with survival rates at 12, 18, and 24 mo of 70%, 36%, and 25%. PK analysis indicated dose-proportional exposure for HCQ. Significant therapy-associated increases in AV and LC3-II were observed in PBMC and correlated with higher HCQ exposure. These data establish that autophagy inhibition is achievable with HCQ, but dose-limiting toxicity prevented escalation to higher doses of HCQ. At HCQ 600 mg/d, autophagy inhibition was not consistently achieved in patients treated with this regimen, and no significant improvement in overall survival was observed. Therefore, a definitive test of the role of autophagy inhibition in the adjuvant setting for glioma patients awaits the development of lower-toxicity compounds that can achieve more consistent inhibition of autophagy than HCQ.

Combined MTOR and autophagy inhibition: Phase I trial of hydroxychloroquine and temsirolimus in patients with advanced solid tumors and melanoma

Abstract: The combination of temsirolimus (TEM), an MTOR inhibitor, and hydroxychloroquine (HCQ), an autophagy inhibitor, augments cell death in preclinical models. This phase 1 dose-escalation study evaluated the maximum tolerated dose (MTD), safety, preliminary activity, pharmacokinetics, and pharmacodynamics of HCQ in combination with TEM in cancer patients. In the dose escalation portion, 27 patients with advanced solid malignancies were enrolled, followed by a cohort expansion at the top dose level in 12 patients with metastatic melanoma. The combination of HCQ and TEM was well tolerated, and grade 3 or 4 toxicity was limited to anorexia (7%), fatigue (7%), and nausea (7%). An MTD was not reached for HCQ, and the recommended phase II dose was HCQ 600 mg twice daily in combination with TEM 25 mg weekly. Other common grade 1 or 2 toxicities included fatigue, anorexia, nausea, stomatitis, rash, and weight loss. No responses were observed; however, 14/21 (67%) patients in the dose escalation and 14/19 (74%) patients with melanoma achieved stable disease. The median progression-free survival in 13 melanoma patients treated with HCQ 1200mg/d in combination with TEM was 3.5 mo. Novel 18-fluorodeoxyglucose positron emission tomography (FDG-PET) measurements predicted clinical outcome and provided further evidence that the addition of HCQ to TEM produced metabolic stress on tumors in patients that experienced clinical benefit. Pharmacodynamic evidence of autophagy inhibition was evident in serial PBMC and tumor biopsies only in patients treated with 1200 mg daily HCQ. This study indicates that TEM and HCQ is safe and tolerable, modulates autophagy in patients, and has significant antitumor activity. Further studies combining MTOR and autophagy inhibitors in cancer patients are warranted.

Dysregulated autophagy in the RPE is associated with increased susceptibility to oxidative stress and AMD.

Abstract: Autophagic dysregulation has been suggested in a broad range of neurodegenerative diseases including age-related macular degeneration (AMD). To test whether the autophagy pathway plays a critical role to protect retinal pigmented epithelial (RPE) cells against oxidative stress, we exposed ARPE-19 and primary cultured human RPE cells to both acute (3 and 24 h) and chronic (14 d) oxidative stress and monitored autophagy by western blot, PCR, and autophagosome counts in the presence or absence of autophagy modulators. Acute oxidative stress led to a marked increase in autophagy in the RPE, whereas autophagy was reduced under chronic oxidative stress. Upregulation of autophagy by rapamycin decreased oxidative stress-induced generation of reactive oxygen species (ROS), whereas inhibition of autophagy by 3-methyladenine (3-MA) or by knockdown of ATG7 or BECN1 increased ROS generation, exacerbated oxidative stress-induced reduction of mitochondrial activity, reduced cell viability, and increased lipofuscin. Exam...

Expression of the autophagy substrate SQSTM1/p62 is restored during prolonged starvation depending on transcriptional upregulation and autophagy-derived amino acids.

Abstract: SQSTM1/p62 (sequestosome 1) is a multifunctional signaling molecule, involved in a variety of cellular pathways. SQSTM1 is one of the best-known autophagic substrates, and is therefore widely used as an indicator of autophagic degradation. Here we report that the expression level of SQSTM1 can be restored during prolonged starvation. Upon starvation, SQSTM1 is initially degraded by autophagy. However, SQSTM1 is restored back to basal levels during prolonged starvation in mouse embryonic fibroblasts and HepG2 cells, but not in HeLa and HEK293 cells. Restoration of SQSTM1 depends on its transcriptional upregulation, which is triggered by amino acid starvation. Furthermore, amino acids derived from the autophagy-lysosome pathway are used for de novo synthesis of SQSTM1 under starvation conditions. The restoration of SQSTM1 is independent of reactivation of MTORC1 (mechanistic target of rapamycin complex 1). These results suggest that the expression level of SQSTM1 in starved cells is determined by at least 3 factors: autophagic degradation, transcriptional upregulation, and availability of lysosomal-derived amino acids. The results of this study also indicate that the expression level of SQSTM1 does not always inversely correlate with autophagic activity.

Phase I trial of hydroxychloroquine with dose-intense temozolomide in patients with advanced solid tumors and melanoma

Abstract: Blocking autophagy with hydroxychloroquine (HCQ) augments cell death associated with alkylating chemotherapy in preclinical models. This phase I study evaluated the maximum tolerated dose (MTD), safety, preliminary activity, pharmacokinetics, and pharmacodynamics of HCQ in combination with dose-intense temozolomide (TMZ) in patients with advanced solid malignancies. Forty patients (73% metastatic melanoma) were treated with oral HCQ 200 to 1200 mg daily with dose-intense oral TMZ 150 mg/m2 daily for 7/14 d. This combination was well tolerated with no recurrent dose-limiting toxicities observed. An MTD was not reached for HCQ and the recommended phase II dose was HCQ 600 mg twice daily combined with dose-intense TMZ. Common toxicities included grade 2 fatigue (55%), anorexia (28%), nausea (48%), constipation (20%), and diarrhea (20%). Partial responses and stable disease were observed in 3/22 (14%) and 6/22 (27%) patients with metastatic melanoma. In the final dose cohort 2/6 patients with refractory BRAF ...

Combined autophagy and proteasome inhibition: a phase 1 trial of hydroxychloroquine and bortezomib in patients with relapsed/refractory myeloma.

Abstract: The efficacy of proteasome inhibition for myeloma is limited by therapeutic resistance, which may be mediated by activation of the autophagy pathway as an alternative mechanism of protein degradation. Preclinical studies demonstrate that autophagy inhibition with hydroxychloroquine augments the antimyeloma efficacy of the proteasome inhibitor bortezomib. We conducted a phase I trial combining bortezomib and hydroxychloroquine for relapsed or refractory myeloma. We enrolled 25 patients, including 11 (44%) refractory to prior bortezomib. No protocol-defined dose-limiting toxicities occurred, and we identified a recommended phase 2 dose of hydroxychloroquine 600 mg twice daily with standard doses of bortezomib, at which we observed dose-related gastrointestinal toxicity and cytopenias. Of 22 patients evaluable for response, 3 (14%) had very good partial responses, 3 (14%) had minor responses, and 10 (45%) had a period of stable disease. Electron micrographs of bone marrow plasma cells collected at baseline, ...

Autophagy in osteoblasts is involved in mineralization and bone homeostasis.

Abstract: Bone remodeling is a tightly controlled mechanism in which osteoblasts (OB), the cells responsible for bone formation, osteoclasts (OC), the cells specialized for bone resorption, and osteocytes, the multifunctional mechanosensing cells embedded in the bone matrix, are the main actors. Increased oxidative stress in OB, the cells producing and mineralizing bone matrix, has been associated with osteoporosis development but the role of autophagy in OB has not yet been addressed. This is the goal of the present study. We first show that the autophagic process is induced in OB during mineralization. Then, using knockdown of autophagy-essential genes and OB-specific autophagy-deficient mice, we demonstrate that autophagy deficiency reduces mineralization capacity. Moreover, our data suggest that autophagic vacuoles could be used as vehicles in OB to secrete apatite crystals. In addition, autophagy-deficient OB exhibit increased oxidative stress and secretion of the receptor activator of NFKB1 (TNFSF11/RANKL), favoring generation of OC, the cells specialized in bone resorption. In vivo, we observed a 50% reduction in trabecular bone mass in OB-specific autophagy-deficient mice. Taken together, our results show for the first time that autophagy in OB is involved both in the mineralization process and in bone homeostasis. These findings are of importance for mineralized tissues which extend from corals to vertebrates and uncover new therapeutic targets for calcified tissue-related metabolic pathologies.

Autophagy in microglia degrades extracellular β-amyloid fibrils and regulates the NLRP3 inflammasome

Abstract: Accumulation of β-amyloid (Aβ) and resultant inflammation are critical pathological features of Alzheimer disease (AD). Microglia, a primary immune cell in brain, ingests and degrades extracellular Aβ fibrils via the lysosomal system. Autophagy is a catabolic process that degrades native cellular components, however, the role of autophagy in Aβ degradation by microglia and its effects on AD are unknown. Here we demonstrate a novel role for autophagy in the clearance of extracellular Aβ fibrils by microglia and in the regulation of the Aβ-induced NLRP3 (NLR family, pyrin domain containing 3) inflammasome using microglia specific atg7 knockout mice and cell cultures. We found in microglial cultures that Aβ interacts with MAP1LC3B-II via OPTN/optineurin and is degraded by an autophagic process mediated by the PRKAA1 pathway. We anticipate that enhancing microglial autophagy may be a promising new therapeutic strategy for AD.

Combined autophagy and HDAC inhibition: a phase I safety, tolerability, pharmacokinetic, and pharmacodynamic analysis of hydroxychloroquine in combination with the HDAC inhibitor vorinostat in patients with advanced solid tumors.

Abstract: We previously reported that inhibition of autophagy significantly augmented the anticancer activity of the histone deacetylase (HDAC) inhibitor vorinostat (VOR) through a cathepsin D-mediated mechanism. We thus conducted a first-in-human study to investigate the safety, preliminary efficacy, pharmacokinetics (PK), and pharmacodynamics (PD) of the combination of the autophagy inhibitor hydroxychloroquine (HCQ) and VOR in patients with advanced solid tumors. Of 27 patients treated in the study, 24 were considered fully evaluable for study assessments and toxicity. Patients were treated orally with escalating doses of HCQ daily (QD) (d 2 to 21 of a 21-d cycle) in combination with 400 mg VOR QD (d one to 21). Treatment-related adverse events (AE) included grade 1 to 2 nausea, diarrhea, fatigue, weight loss, anemia, and elevated creatinine. Grade 3 fatigue and/or myelosuppression were observed in a minority of patients. Fatigue and gastrointestinal AE were dose-limiting toxicities. Six-hundred milligrams HCQ a...

Impaired autophagy flux is associated with neuronal cell death after traumatic brain injury.

Abstract: Dysregulation of autophagy contributes to neuronal cell death in several neurodegenerative and lysosomal storage diseases. Markers of autophagy are also increased after traumatic brain injury (TBI), but its mechanisms and function are not known. Following controlled cortical impact (CCI) brain injury in GFP-Lc3 (green fluorescent protein-LC3) transgenic mice, we observed accumulation of autophagosomes in ipsilateral cortex and hippocampus between 1 and 7 d. This accumulation was not due to increased initiation of autophagy but rather to a decrease in clearance of autophagosomes, as reflected by accumulation of the autophagic substrate SQSTM1/p62 (sequestosome 1). This was confirmed by ex vivo studies, which demonstrated impaired autophagic flux in brain slices from injured as compared to control animals. Increased SQSTM1 peaked at d 1-3 but resolved by d 7, suggesting that the defect in autophagy flux is temporary. The early impairment of autophagy is at least in part caused by lysosomal dysfunction, as evidenced by lower protein levels and enzymatic activity of CTSD (cathepsin D). Furthermore, immediately after injury both autophagosomes and SQSTM1 accumulated predominantly in neurons. This was accompanied by appearance of SQSTM1 and ubiquitin-positive puncta in the affected cells, suggesting that, similar to the situation observed in neurodegenerative diseases, impaired autophagy may contribute to neuronal injury. Consistently, GFP-LC3 and SQSTM1 colocalized with markers of both caspase-dependent and caspase-independent cell death in neuronal cells proximal to the injury site. Taken together, our data indicated for the first time that autophagic clearance is impaired early after TBI due to lysosomal dysfunction, and correlates with neuronal cell death.

Coronavirus NSP6 restricts autophagosome expansion

Abstract: Autophagy is a cellular response to starvation that generates autophagosomes to carry long-lived proteins and cellular organelles to lysosomes for degradation. Activation of autophagy by viruses can provide an innate defense against infection, and for (+) strand RNA viruses autophagosomes can facilitate assembly of replicase proteins. We demonstrated that nonstructural protein (NSP) 6 of the avian coronavirus, infectious bronchitis virus (IBV), generates autophagosomes from the ER. A statistical analysis of MAP1LC3B puncta showed that NSP6 induced greater numbers of autophagosomes per cell compared with starvation, but the autophagosomes induced by NSP6 had smaller diameters compared with starvation controls. Small diameter autophagosomes were also induced by infection of cells with IBV, and by NSP6 proteins of MHV and SARS and NSP5, NSP6, and NSP7 of arterivirus PRRSV. Analysis of WIPI2 puncta induced by NSP6 suggests that NSP6 limits autophagosome diameter at the point of omegasome formation. IBV NSP6 also limited autophagosome and omegasome expansion in response to starvation and Torin1 and could therefore limit the size of autophagosomes induced following inhibition of MTOR signaling, as well as those induced independently by the NSP6 protein itself. MAP1LC3B-puncta induced by NSP6 contained SQSTM1, which suggests they can incorporate autophagy cargos. However, NSP6 inhibited the autophagosome/lysosome expansion normally seen following starvation. Taken together the results show that coronavirus NSP6 proteins limit autophagosome expansion, whether they are induced directly by the NSP6 protein, or indirectly by starvation or chemical inhibition of MTOR signaling. This may favor coronavirus infection by compromising the ability of autophagosomes to deliver viral components to lysosomes for degradation.

Mesenchymal stem cells enhance autophagy and increase β-amyloid clearance in Alzheimer disease models

Abstract: Current evidence suggests a central role for autophagy in Alzheimer disease (AD), and dysfunction in the autophagic system may lead to amyloid-β (Aβ) accumulation. Using in vitro and in vivo AD models, the present study investigated whether mesenchymal stem cells (MSCs) could enhance autophagy and thus exert a neuroprotective effect through modulation of Aβ clearance In Aβ-treated neuronal cells, MSCs increased cellular viability and enhanced LC3-II expression compared with cells treated with Aβ only. Immunofluorescence revealed that MSC coculture in Aβ-treated neuronal cells increased the number of LC3-II-positive autophagosomes that were colocalized with a lysosomal marker. Ultrastructural analysis revealed that most autophagic vacuoles (AVs) in Aβ-treated cells were not fused with lysosomes, whereas a large portion of autophagosomes were conjoined with lysosomes in MSCs cocultured with Aβ-treated neuronal cells. Furthermore, MSC coculture markedly increased Aβ immunoreactivity colocalized within lysosomes and decreased intracellular Aβ levels compared with Aβ-treated cells. In Aβ-treated animals, MSC administration significantly increased autophagosome induction, final maturation of late AVs, and fusion with lysosomes. Moreover, MSC administration significantly reduced the level of Aβ in the hippocampus, which was elevated in Aβ-treated mice, concomitant with increased survival of hippocampal neurons. Finally, MSC coculture upregulated BECN1/Beclin 1 expression in AD models. These results suggest that MSCs significantly enhance autolysosome formation and clearance of Aβ in AD models, which may lead to increased neuronal survival against Aβ toxicity. Modulation of the autophagy pathway to repair the damaged AD brain using MSCs would have a significant impact on future strategies for AD treatment.

Defining and measuring autophagosome flux—concept and reality

Abstract: The autophagic system is involved in both bulk degradation of primarily long-lived cytoplasmic proteins as well as in selective degradation of cytoplasmic organelles. Autophagic flux is often defined as a measure of autophagic degradation activity, and a number of methods are currently utilized to assess autophagic flux. However, despite major advances in measuring various molecular aspects of the autophagic machinery, we remain less able to express autophagic flux in a highly sensitive, robust, and well-quantifiable manner. Here, we describe a conceptual framework for defining and measuring autophagosome flux at the single-cell level. The concept discussed here is based on the theoretical framework of metabolic control analysis, which distinguishes between the pathway along which there is a flow of material and the quantitative measure of this flow. By treating the autophagic system as a multistep pathway with each step characterized by a particular rate, we are able to provide a single-cell fluorescence live-cell imaging-based approach that describes the accurate assessment of the complete autophagosome pool size, the autophagosome flux, and the transition time required to turn over the intracellular autophagosome pool. In doing so, this perspective provides clarity on whether the system is at steady state or in a transient state moving towards a new steady state. It is hoped that this theoretical account of quantitatively measuring autophagosome flux may contribute towards a new direction in the field of autophagy, a standardized approach that allows the establishment of systematic flux databases of clinically relevant cell and tissue types that serve as important model systems for human pathologies.

MTOR-independent, autophagic enhancer trehalose prolongs motor neuron survival and ameliorates the autophagic flux defect in a mouse model of amyotrophic lateral sclerosis

Abstract: Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder caused by selective motor neuron degeneration. Abnormal protein aggregation and impaired protein degradation pathways may contribute to the disease pathogenesis. Although it has been reported that autophagy is altered in patients and animal model of ALS, little is known about the role of autophagy in motor neuron degeneration in this disease. Our previous study shows that rapamycin, an MTOR-dependent autophagic activator, accelerates disease progression in the SOD1G93A mouse model of ALS. In the present report, we have assessed the role of the MTOR-independent autophagic pathway in ALS by determining the effect of the MTOR-independent autophagic inducer trehalose on disease onset and progression, and on motor neuron degeneration in SOD1G93A mice. We have found that trehalose significantly delays disease onset prolongs life span, and reduces motor neuron loss in the spinal cord of SOD1G93A mice. Most importantly, we have docume...

Inhibition of autophagy enhances the anticancer activity of silver nanoparticles

Abstract: Silver nanoparticles (Ag NPs) are cytotoxic to cancer cells and possess excellent potential as an antitumor agent. A variety of nanoparticles have been shown to induce autophagy, a critical cellular degradation process, and the elevated autophagy in most of these situations promotes cell death. Whether Ag NPs can induce autophagy and how it might affect the anticancer activity of Ag NPs has not been reported. Here we show that Ag NPs induced autophagy in cancer cells by activating the PtdIns3K signaling pathway. The autophagy induced by Ag NPs was characterized by enhanced autophagosome formation, normal cargo degradation, and no disruption of lysosomal function. Consistent with these properties, the autophagy induced by Ag NPs promoted cell survival, as inhibition of autophagy by either chemical inhibitors or ATG5 siRNA enhanced Ag NPs-elicited cancer cell killing. We further demonstrated that wortmannin, a widely used inhibitor of autophagy, significantly enhanced the antitumor effect of Ag NPs in the B16 mouse melanoma cell model. Our results revealed a novel biological activity of Ag NPs in inducing cytoprotective autophagy, and inhibition of autophagy may be a useful strategy for improving the efficacy of Ag NPs in anticancer therapy.

Small molecule-driven mitophagy-mediated NLRP3 inflammasome inhibition is responsible for the prevention of colitis-associated cancer

Abstract: Nonresolving inflammation in the intestine predisposes individuals to the development of colitis-associated cancer (CAC). Inflammasomes are thought to mediate intestinal homeostasis, and their dysregulation contributes to inflammatory bowel diseases and CAC. However, few agents have been reported to reduce CAC by targeting inflammasomes. Here we show that the small molecule andrographolide (Andro) protects mice against azoxymethane/dextran sulfate sodium-induced colon carcinogenesis through inhibiting the NLRP3 inflammasome. Administration of Andro significantly attenuated colitis progression and tumor burden. Andro also inhibited NLRP3 inflammasome activation in macrophages both in vivo and in vitro, as indicated by reduced expression of cleaved CASP1, disruption of NLRP3-PYCARD-CASP1 complex assembly, and lower IL1B secretion. Importantly, Andro was found to trigger mitophagy in macrophages, leading to a reversed mitochondrial membrane potential collapse, which in turn inactivated the NLRP3 inflammasome. Moreover, downregulation of the PIK3CA-AKT1-MTOR-RPS6KB1 pathway accounted for Andro-induced autophagy. Finally, Andro-driven inhibition of the NLRP3 inflammasome and amelioration of murine models for colitis and CAC were significantly blocked by BECN1 knockdown, or by various autophagy inhibitors. Taken together, our findings demonstrate that mitophagy-mediated NLRP3 inflammasome inhibition by Andro is responsible for the prevention of CAC. Our data may help guide decisions regarding the use of Andro in patients with inflammatory bowel diseases, which ultimately reduces the risk of CAC.

A novel ATG4B antagonist inhibits autophagy and has a negative impact on osteosarcoma tumors

Abstract: Autophagy has been implicated in the progression and chemoresistance of various cancers. In this study, we have shown that osteosarcoma Saos-2 cells lacking ATG4B, a cysteine proteinase that activates LC3B, are defective in autophagy and fail to form tumors in mouse models. By combining in silico docking with in vitro and cell-based assays, we identified small compounds that suppressed starvation-induced protein degradation, LC3B lipidation, and formation of autophagic vacuoles. NSC185058 effectively inhibited ATG4B activity in vitro and in cells while having no effect on MTOR and PtdIns3K activities. In addition, this ATG4B antagonist had a negative impact on the development of Saos-2 osteosarcoma tumors in vivo. We concluded that tumor suppression was due to a reduction in ATG4B activity, since we found autophagy suppressed within treated tumors and the compound had no effects on oncogenic protein kinases. Our findings demonstrate that ATG4B is a suitable anti-autophagy target and a promising therapeutic target to treat osteosarcoma.

Endoplasmic reticulum stress induced by tunicamycin and thapsigargin protects against transient ischemic brain injury: Involvement of PARK2-dependent mitophagy

Abstract: Transient cerebral ischemia leads to endoplasmic reticulum (ER) stress. However, the contributions of ER stress to cerebral ischemia are not clear. To address this issue, the ER stress activators tunicamycin (TM) and thapsigargin (TG) were administered to transient middle cerebral artery occluded (tMCAO) mice and oxygen-glucose deprivation-reperfusion (OGD-Rep.)-treated neurons. Both TM and TG showed significant protection against ischemia-induced brain injury, as revealed by reduced brain infarct volume and increased glucose uptake rate in ischemic tissue. In OGD-Rep.-treated neurons, 4-PBA, the ER stress releasing mechanism, counteracted the neuronal protection of TM and TG, which also supports a protective role of ER stress in transient brain ischemia. Knocking down the ER stress sensor Eif2s1, which is further activated by TM and TG, reduced the OGD-Rep.-induced neuronal cell death. In addition, both TM and TG prevented PARK2 loss, promoted its recruitment to mitochondria, and activated mitophagy during reperfusion after ischemia. The neuroprotection of TM and TG was reversed by autophagy inhibition (3-methyladenine and Atg7 knockdown) as well as Park2 silencing. The neuroprotection was also diminished in Park2+/− mice. Moreover, Eif2s1 and downstream Atf4 silencing reduced PARK2 expression, impaired mitophagy induction, and counteracted the neuroprotection. Taken together, the present investigation demonstrates that the ER stress induced by TM and TG protects against the transient ischemic brain injury. The PARK2-mediated mitophagy may be underlying the protection of ER stress. These findings may provide a new strategy to rescue ischemic brains by inducing mitophagy through ER stress activation.

Autophagy modulates SNCA/α-synuclein release, thereby generating a hostile microenvironment

Abstract: SNCA/α-synuclein aggregation plays a crucial role in synucleinopathies such as Parkinson disease and dementia with Lewy bodies. Aggregating and nonaggregating SNCA species are degraded by the autophagy-lysosomal pathway (ALP). Previously, we have shown that the ALP is not only responsible for SNCA degradation but is also involved in the intracellular aggregation process of SNCA. An additional role of extracellular SNCA in the pathology of synucleinopathies substantiating a prion-like propagation hypothesis has been suggested since released SNCA species and spreading of SNCA pathology throughout neural cells have been observed. However, the molecular interplay between intracellular pathways, SNCA aggregation, release, and response of the local microenvironment remains unknown. Here, we attributed SNCA-induced toxicity mainly to secreted species in a cell culture model of SNCA aggregation and in SNCA transgenic mice: We showed that ALP inhibition by bafilomycinA1 reduced intracellular SNCA aggregation but increased secretion of smaller oligomers that exacerbated microenvironmental response including uptake, inflammation, and cellular damage. Low-aggregated SNCA was predominantly released by exosomes and RAB11A-associated pathways whereas high-aggregated SNCA was secreted by membrane shedding. In summary, our study revealed a novel role of the ALP by linking protein degradation to nonclassical secretion for toxic SNCA species. Thus, impaired ALP in the diseased brain not only limits intracellular degradation of misfolded proteins, but also leads to a detrimental microenvironmental response due to enhanced SNCA secretion. These findings suggest that the major toxic role of SNCA is related to its extracellular species and further supports a protective role of intracellular SNCA aggregation.

Autophagosomes, phagosomes, autolysosomes, phagolysosomes, autophagolysosomes... wait, I'm confused.

Abstract: When an autophagosome or an amphisome fuse with a lysosome, the resulting compartment is referred to as an autolysosome Some people writing papers on the topic of autophagy use the terms “autolysosome” and “autophagolysosome” interchangeably We contend that these words should be used to denote 2 different compartments, and that it is worthwhile maintaining this distinction—the autophagolysosome has a particular origin in the process of xenophagy that makes it distinct from an autolysosome

Acidic extracellular pH neutralizes the autophagy-inhibiting activity of chloroquine: Implications for cancer therapies

Abstract: Acidic pH is an important feature of tumor microenvironment and a major determinant of tumor progression. We reported that cancer cells upregulate autophagy as a survival mechanism to acidic stress. Inhibition of autophagy by administration of chloroquine (CQ) in combination anticancer therapies is currently evaluated in clinical trials. We observed in 3 different human cancer cell lines cultured at acidic pH that autophagic flux is not blocked by CQ. This was consistent with a complete resistance to CQ toxicity in cells cultured in acidic conditions. Conversely, the autophagy-inhibiting activity of Lys-01, a novel CQ derivative, was still detectable at low pH. The lack of CQ activity was likely dependent on a dramatically reduced cellular uptake at acidic pH. Using cell lines stably adapted to chronic acidosis we could confirm that CQ lack of activity was merely caused by acidic pH. Moreover, unlike CQ, Lys-01 was able to kill low pH-adapted cell lines, although higher concentrations were required as compared with cells cultured at normal pH conditions. Notably, buffering medium pH in low pH-adapted cell lines reverted CQ resistance. In vivo analysis of tumors treated with CQ showed that accumulation of strong LC3 signals was observed only in normoxic areas but not in hypoxic/acidic regions. Our observations suggest that targeting autophagy in the tumor environment by CQ may be limited to well-perfused regions but not achieved in acidic regions, predicting possible limitations in efficacy of CQ in antitumor therapies.

iLIR: A web resource for prediction of Atg8-family interacting proteins

Abstract: Macroautophagy was initially considered to be a nonselective process for bulk breakdown of cytosolic material. However, recent evidence points toward a selective mode of autophagy mediated by the so-called selective autophagy receptors (SARs). SARs act by recognizing and sorting diverse cargo substrates (e.g., proteins, organelles, pathogens) to the autophagic machinery. Known SARs are characterized by a short linear sequence motif (LIR-, LRS-, or AIM-motif) responsible for the interaction between SARs and proteins of the Atg8 family. Interestingly, many LIR-containing proteins (LIRCPs) are also involved in autophagosome formation and maturation and a few of them in regulating signaling pathways. Despite recent research efforts to experimentally identify LIRCPs, only a few dozen of this class of—often unrelated—proteins have been characterized so far using tedious cell biological, biochemical, and crystallographic approaches. The availability of an ever-increasing number of complete eukaryotic genomes provides a grand challenge for characterizing novel LIRCPs throughout the eukaryotes. Along these lines, we developed iLIR, a freely available web resource, which provides in silico tools for assisting the identification of novel LIRCPs. Given an amino acid sequence as input, iLIR searches for instances of short sequences compliant with a refined sensitive regular expression pattern of the extended LIR motif (xLIR-motif) and retrieves characterized protein domains from the SMART database for the query. Additionally, iLIR scores xLIRs against a custom position-specific scoring matrix (PSSM) and identifies potentially disordered subsequences with protein interaction potential overlapping with detected xLIR-motifs. Here we demonstrate that proteins satisfying these criteria make good LIRCP candidates for further experimental verification. Domain architecture is displayed in an informative graphic, and detailed results are also available in tabular form. We anticipate that iLIR will assist with elucidating the full complement of LIRCPs in eukaryotes.

Autophagy facilitates TLR4- and TLR3-triggered migration and invasion of lung cancer cells through the promotion of TRAF6 ubiquitination

Abstract: Autophagy contributes to the pathogenesis of cancer, whereas toll-like receptors (TLRs) also play an important role in cancer development and immune escape. However, little is known about the potential interaction between TLR signaling and autophagy in cancer cells. Here we show that autophagy induced by TLR4 or TLR3 activation enhances various cytokine productions through promoting TRAF6 (TNF receptor-associated factor 6, E3 ubiquitin protein ligase) ubiquitination and thus facilitates migration and invasion of lung cancer cells. Stimulation of TLR4 and TLR3 with lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid [poly(I:C)] respectively triggered autophagy in lung cancer cells. This was mediated by the adaptor protein, toll-like receptor adaptor molecule 1 (TICAM1/TRIF), and was required for TLR4- and TLR3-induced increases in the production of IL6, CCL2/MCP-1 [chemokine (C-C motif) ligand 2], CCL20/MIP-3α [chemokine (C-C motif) ligand 20], VEGFA (vascular endothelial growth factor A), and MMP2 [matrix metallopeptidase 2 (gelatinase A, 72 kDa gelatinase, 72 kDa type IV collagenase)]. These cytokines appeared to be necessary for enhanced migration and invasion of lung cancer cells upon TLR activation. Remarkably, inhibition of autophagy by chemical or genetic approaches blocked TLR4- or TLR3-induced Lys63 (K63)-linked ubiquitination of TRAF6 that was essential for activation of MAPK and NFKB (nuclear factor of kappa light polypeptide gene enhancer in B-cells) pathways, both of which were involved in the increased production of the cytokines. Collectively, these results identify induction of autophagy by TLR4 and TLR3 as an important mechanism that drives lung cancer progression, and indicate that inhibition of autophagy may be a useful strategy in the treatment of lung cancer.

Hypoxia-induced MIR155 is a potent autophagy inducer by targeting multiple players in the MTOR pathway

Abstract: Hypoxia activates autophagy, an evolutionarily conserved cellular catabolic process. Dysfunction in the autophagy pathway has been implicated in an increasing number of human diseases, including cancer. Hypoxia induces upregulation of a specific set of microRNAs (miRNAs) in a variety of cell types. Here, we describe hypoxia-induced MIR155 as a potent inducer of autophagy. Enforced expression of MIR155 increases autophagic activity in human nasopharyngeal cancer and cervical cancer cells. Knocking down endogenous MIR155 inhibits hypoxia-induced autophagy. We demonstrated that MIR155 targets multiple players in MTOR signaling, including RHEB, RICTOR, and RPS6KB2. MIR155 suppresses target-gene expression by directly interacting with their 3′ untranslated regions (UTRs), mutations of the binding sites abolish their MIR155 responsiveness. Furthermore, by downregulating MTOR signaling, MIR155 also attenuates cell proliferation and induces G1/S cell cycle arrest. Collectively, these data present a new role for M...

Autophagy promotes resistance to photodynamic therapy-induced apoptosis selectively in colorectal cancer stem-like cells

Abstract: Recent studies have indicated that cancer stem-like cells (CSCs) exhibit a high resistance to current therapeutic strategies, including photodynamic therapy (PDT), leading to the recurrence and progression of colorectal cancer (CRC). In cancer, autophagy acts as both a tumor suppressor and a tumor promoter. However, the role of autophagy in the resistance of CSCs to PDT has not been reported. In this study, CSCs were isolated from colorectal cancer cells using PROM1/CD133 (prominin 1) expression, which is a surface marker commonly found on stem cells of various tissues. We demonstrated that PpIX-mediated PDT induced the formation of autophagosomes in PROM1/CD133(+) cells, accompanied by the upregulation of autophagy-related proteins ATG3, ATG5, ATG7, and ATG12. The inhibition of PDT-induced autophagy by pharmacological inhibitors and silencing of the ATG5 gene substantially triggered apoptosis of PROM1/CD133(+) cells and decreased the ability of colonosphere formation in vitro and tumorigenicity in vivo. In conclusion, our results revealed a protective role played by autophagy against PDT in CSCs and indicated that targeting autophagy could be used to elevate the PDT sensitivity of CSCs. These findings would aid in the development of novel therapeutic approaches for CSC treatment.

OsATG7 is required for autophagy-dependent lipid metabolism in rice postmeiotic anther development

Abstract: In flowering plants, the tapetum, the innermost layer of the anther, provides both nutrient and lipid components to developing microspores, pollen grains, and the pollen coat. Though the programmed cell death of the tapetum is one of the most critical and sensitive steps for fertility and is affected by various environmental stresses, its regulatory mechanisms remain mostly unknown. Here we show that autophagy is required for the metabolic regulation and nutrient supply in anthers and that autophagic degradation within tapetum cells is essential for postmeiotic anther development in rice. Autophagosome-like structures and several vacuole-enclosed lipid bodies were observed in postmeiotic tapetum cells specifically at the uninucleate stage during pollen development, which were completely abolished in a retrotransposon-insertional OsATG7 (autophagy-related 7)-knockout mutant defective in autophagy, suggesting that autophagy is induced in tapetum cells. Surprisingly, the mutant showed complete sporophytic ma...

TSPO interacts with VDAC1 and triggers a ROS-mediated inhibition of mitochondrial quality control

Abstract: The 18-kDa TSPO (translocator protein) localizes on the outer mitochondrial membrane (OMM) and participates in cholesterol transport. Here, we report that TSPO inhibits mitochondrial autophagy downstream of the PINK1-PARK2 pathway, preventing essential ubiquitination of proteins. TSPO abolishes mitochondrial relocation of SQSTM1/p62 (sequestosome 1), and consequently that of the autophagic marker LC3 (microtubule-associated protein 1 light chain 3), thus leading to an accumulation of dysfunctional mitochondria, altering the appearance of the network. Independent of cholesterol regulation, the modulation of mitophagy by TSPO is instead dependent on VDAC1 (voltage-dependent anion channel 1), to which TSPO binds, reducing mitochondrial coupling and promoting an overproduction of reactive oxygen species (ROS) that counteracts PARK2-mediated ubiquitination of proteins. These data identify TSPO as a novel element in the regulation of mitochondrial quality control by autophagy, and demonstrate the importance for cell homeostasis of its expression ratio with VDAC1.

Phase I clinical trial and pharmacodynamic evaluation of combination hydroxychloroquine and doxorubicin treatment in pet dogs treated for spontaneously occurring lymphoma

Abstract: Autophagy is a lysosomal degradation process that may act as a mechanism of survival in a variety of cancers. While pharmacologic inhibition of autophagy with hydroxychloroquine (HCQ) is currently being explored in human clinical trials, it has never been evaluated in canine cancers. Non-Hodgkin lymphoma (NHL) is one of the most prevalent tumor types in dogs and has similar pathogenesis and response to treatment as human NHL. Clinical trials in canine patients are conducted in the same way as in human patients, thus, to determine a maximum dose of HCQ that can be combined with a standard chemotherapy, a Phase I, single arm, dose escalation trial was conducted in dogs with spontaneous NHL presenting as patients to an academic, tertiary-care veterinary teaching hospital. HCQ was administered daily by mouth throughout the trial, beginning 72 h prior to doxorubicin (DOX), which was given intravenously on a 21-d cycle. Peripheral blood mononuclear cells and biopsies were collected before and 3 d after HCQ treatment and assessed for autophagy inhibition and HCQ concentration. A total of 30 patients were enrolled in the trial. HCQ alone was well tolerated with only mild lethargy and gastrointestinal-related adverse events. The overall response rate (ORR) for dogs with lymphoma was 93.3%, with median progression-free interval (PFI) of 5 mo. Pharmacokinetic analysis revealed a 100-fold increase in HCQ in tumors compared with plasma. There was a trend that supported therapy-induced increase in LC3-II (the cleaved and lipidated form of microtubule-associated protein 1 light chain 3/LC3, which serves as a maker for autophagosomes) and SQSTM1/p62 (sequestosome 1) after treatment. The superior ORR and comparable PFI to single-agent DOX provide strong support for further evaluation via randomized, placebo-controlled trials in canine and human NHL.

Itraconazole suppresses the growth of glioblastoma through induction of autophagy: Involvement of abnormal cholesterol trafficking

Abstract: Glioblastoma is one of the most aggressive human cancers with poor prognosis, and therefore a critical need exists for novel therapeutic strategies for management of glioblastoma patients. Itraconazole, a traditional antifungal drug, has been identified as a novel potential anticancer agent due to its inhibitory effects on cell proliferation and tumor angiogenesis; however, the molecular mechanisms involved are still unclear. Here, we show that itraconazole inhibits the proliferation of glioblastoma cells both in vitro and in vivo. Notably, we demonstrate that treatment with itraconazole induces autophagic progression in glioblastoma cells, while blockage of autophagy markedly reverses the antiproliferative activities of itraconazole, suggesting an antitumor effect of autophagy in response to itraconazole treatment. Functional studies revealed that itraconazole retarded the trafficking of cholesterol from late endosomes and lysosomes to the plasma membrane by reducing the levels of SCP2, resulting in repr...

Boning up on autophagy: the role of autophagy in skeletal biology.

Abstract: From an evolutionary perspective, the major function of bone is to provide stable sites for muscle attachment and affording protection of vital organs, especially the heart and lungs (ribs) and spinal cord (vertebrae and intervertebral discs) However, bone has a considerable number of other functions: serving as a store for mineral ions, providing a site for blood cell synthesis and participating in a complex system-wide endocrine system Not surprisingly, bone and cartilage cell homeostasis is tightly controlled, as is the maintenance of tissue structure and mass While a great deal of new information is accruing concerning skeletal cell homeostasis, one relatively new observation is that the cells of bone (osteoclasts osteoblasts and osteocytes) and cartilage (chondrocytes) exhibit autophagy The focus of this review is to examine the significance of this process in terms of the functional demands of the skeleton in health and during growth and to provide evidence that dysregulation of the autophagic r