Role for nanomaterial-autophagy interaction in neurodegenerative disease

Nanotechnology is the control and manipulation of materials in the size range of 1-100 nm. Coupled with increasing research into potential beneficial applications of nanotechnology, there is an urgent need for the study of possible health risks. Several researchers, including those in our laboratory, have demonstrated elevated levels of autophagic vacuoles upon exposure of cells to certain nanomaterials, including carbon- and metal-based nanoparticles. While this apparent increase in autophagic activity may be an appropriate cellular response toward nanomaterial clearance, often the interaction between nanomaterials and the autophagy pathway is disruptive, resulting in severe morphological changes and coincident cell death. Interestingly, epidemiological studies have identified an association between exposure to combustion-derived ambient particles (which are predominantly nanoscale) and neurological conditions with Alzheimer�s and Parkinson�s disease-like pathologies. As impaired autophagy may play an important role in the pathogenesis of these and other diseases, it is intriguing to speculate about the plausible involvement of nanoscale particulates in this process. The interaction of nanomaterials with the autophagy pathway, and the potential negative consequences of resulting autophagy dysfunction, should be explored further.

Addendum to: Stern ST, Zolnik BS, McLeland CB, Clogston J, Zheng J, McNeil SE. Induction of autophagy in porcine kidney cells by quantum dots: A common cellular response to nanomaterials? Toxicol Sci 2008; In press; DOI: 10.1093/toxsci/kfn137.     

Silver nanoparticles regulate autophagy through lysosome injury and cell hypoxia in prostate cancer cells

With the rapid development of nanotechnology, nanomaterials are now being used for cancer treatment. Although studies on the application of silver nanoparticles in cancer treatment are burgeoning, few studies have investigated the toxicology mechanisms of autophagy in cancer cells under exposure to sublethal silver nanoparticles. Here, we clarified the distinct mechanisms of silver nanoparticles for the regulation of autophagy in prostate cancer PC‐3 cells under sublethal exposure. Silver nanoparticle treatment caused lysosome injury, including the decline of lysosomal membrane integrity, decrease of lysosomal quantity, and attenuation of lysosomal protease activity, which resulted in blockage of autophagic flux. In addition, sublethal silver nanoparticle exposure activated AMP‐activated protein kinase/mammalian target of rapamycin‐dependent signaling pathway to modulate autophagy, which resulted from silver nanoparticles‐induced cell hypoxia and energy deficiency. Taken together, the results show that silver nanoparticles could regulate autophagy via lysosome injury and cell hypoxia in PC‐3 cells under sublethal dose exposure. This study will provide an experimental basis for the cancer therapy of nanomaterials.     

A functionalized single-walled carbon nanotube-induced autophagic cell death in human lung cells through Akt�CTSC2-mTOR signaling

Nanoparticles are now emerging as a novel class of autophagy activators. Functionalized single-walled carbon nanotubes (f-SWCNTs) are valuable nanomaterials in many industries. This article is designed to assess the autophagic response for f-SWCNTs exposure in vitro and in vivo. A few types of f-SWCNTs were screened in human lung adenocarcinoma A549 cells for the autophagic response and related pathways in vitro. Formation of autophagosomes and LC3-II upregulation were confirmed on the basis of electron microscopy and LC3 western blotting for COOH-CNT, but not for PABS-CNT and PEG-CNT. MTT assay showed marked increase in cell viability, when COOH-CNT was added to cells in the presence of autophagy inhibitor 3MA, ATG6 or TSC2 siRNA. Consistent with the involvement of the Akt–TSC1/2–mTOR pathway, the phosphorylation levels of mTOR, mTOR''s substrate S6 and Akt were shown significantly decreased in A549 cells on treatment with COOH-CNT using western blotting. What''s more, autophagy inhibitor 3MA significantly reduced the lung edema in vivo. In a word, COOH-CNT induced autophagic cell death in A549 cells through the AKT–TSC2–mTOR pathway and caused acute lung injury in vivo. Inhibition of autophagy significantly reduced COOH-CNT-induced autophagic cell death and ameliorated acute lung injury in mice, suggesting a potential remedy to address the growing concerns on the safety of nanomaterials.     

Rare earth oxide nanocrystals as a new class of autophagy inducers

Functional interaction of nanomaterial with autophagy, a fundamental biological process for cellular degradation, is of great interest to nanobiology. Rare earth nanomaterials hold tremendous potential for a variety of diagnostic and therapeutic applications and have also been reported to protect cells against bacterial, viral and oxidative stress. In a brief communication we report that both light and heavy classes of rare earth oxide nanocrystals (REOs) elicit an autophagic response in HeLa cells, a human cancer cell line, in a dose- and time-dependent manner. The autophagy induced by REOs is complete and is accompanied by vacuolization within the cytoplasm. Autophagy induction may help explain some of the biological effects caused by REOs, and at the same time raises a special point of consideration for this type of nanomaterial, with regard to both safety assessment and application exploration.     

Beclin‐1–p53 interaction is crucial for cell fate determination in embryonal carcinoma cells

Emerging interest on the interrelationship between the apoptotic and autophagy pathways in the context of cancer chemotherapy is providing exciting discoveries. Complexes formed between molecules from both pathways present potential targets for chemotherapeutics design as disruption of such complexes could alter cell survival. This study demonstrates an important role of Beclin‐1 and p53 interaction in cell fate decision of human embryonal carcinoma cells. The findings provide evidence for p53 interaction with Beclin‐1 through the BH3 domain of the latter. This interaction facilitated Beclin‐1 ubiquitination through lysine 48 linkage, resulting in proteasome‐mediated degradation, consequently maintaining a certain constitutive level of Beclin‐1. Disruption of Beclin‐1–p53 interaction through shRNA‐mediated down‐regulation of p53 reduced Beclin‐1 ubiquitination suggesting requirement of p53 for the process. Reduction of ubiquitination consequently resulted in an increase in Beclin‐1 levels with cells showing high autophagic activity. Enforced overexpression of p53 in the p53 down‐regulated cells restored ubiquitination of Beclin‐1 reducing its level and lowering autophagic activity. The Beclin‐1–p53 interaction was also disrupted by exposure to cisplatin‐induced stress resulting in higher level of Beclin‐1 because of lesser ubiquitination. This higher concentration of Beclin‐1 increased autophagy and offered protection to the cells from cisplatin‐induced death. Inhibition of autophagy by either pharmacological or genetic means during cisplatin exposure increased apoptotic death in vitro as well as in xenograft tumours grown in vivo confirming the protective nature of autophagy. Therefore, Beclin‐1–p53 interaction defines one additional molecular subroutine crucial for cell fate decisions in embryonal carcinoma cells.     

Interplay between apoptotic and autophagy pathways after exposure to cerium dioxide nanoparticles in human monocytes

Engineered nanomaterials, defined as having at least one dimension smaller than 100 nm, have revolutionized many technology sectors ranging from therapeutics and diagnostics to environmental monitoring and remediation. This has resulted in a rapid increase in their manufacture over the past few years, accompanied by an increased human exposure potential. However, understanding of the interactions of nanomaterials with biological systems is still rudimentary. We have described that an environmentally and medically relevant nano metal (cerium dioxide) can affect primary human monocyte viability and interact with programmed cell death pathways leading to apoptosis and autophagic cell death. Cerium dioxide nanoparticles (CeO₂ NPs)-induced autophagy acts as a prodeath mechanism and leads to increased cytotoxicity of human monocytes. A better understanding of the implication and biological significance of CeO₂ NPs-induced autophagy and apoptosis will help us understand the risks associated with its uses and develop safer nanomedicine.     

DJ-1 regulation of mitochondrial function and autophagy through oxidative stress

The dysregulation of mitochondrial function has been implicated in the pathogenesis of Parkinson disease.

Mutations in the parkin, PINK1 and DJ-1 genes all result in recessive parkinsonism. Although the protein products of these genes have not been fully characterized, it has been established that all three contribute to the maintenance of mitochondrial function. PINK1 and parkin act in a common pathway to regulate the selective autophagic removal of depolarized mitochondria, but the relationship between DJ-1 and PINK1- and/or parkin-mediated effects on mitochondria and autophagy is less clear. We have shown that loss of DJ-1 leads to mitochondrial phenotypes including reduced membrane potential, increased fragmentation and accumulation of autophagic markers. Supplementing DJ-1-deficient cells with glutathione reverses both mitochondrial and autophagic changes suggesting that DJ-1 may act to maintain mitochondrial function during oxidative stress and thereby alter mitochondrial dynamics and autophagy indirectly.     

DJ-1 regulation of mitochondrial function and autophagy through oxidative stress

The dysregulation of mitochondrial function has been implicated in the pathogenesis of Parkinson disease. Mutations in the parkin, PINK1 and DJ-1 genes all result in recessive parkinsonism. Although the protein products of these genes have not been fully characterized, it has been established that all three contribute to the maintenance of mitochondrial function. PINK1 and parkin act in a common pathway to regulate the selective autophagic removal of depolarized mitochondria, but the relationship between DJ-1 and PINK1- and/or parkin-mediated effects on mitochondria and autophagy is less clear. We have shown that loss of DJ-1 leads to mitochondrial phenotypes including reduced membrane potential, increased fragmentation and accumulation of autophagic markers. Supplementing DJ-1-deficient cells with glutathione reverses both mitochondrial and autophagic changes suggesting that DJ-1 may act to maintain mitochondrial function during oxidative stress and thereby alter mitochondrial dynamics and autophagy indirectly.     

Silencing DJ-1 reveals its contribution in paraquat-induced autophagy

The role of autophagy as a survival strategy of cells constitutes an emerging topic in the study of the pathogenesis of several diseases with autophagic changes being described in a number of age-related neurodegenerative disorders, including Parkinson's disease (PD). Although the etiology of PD is still unknown, both environmental (for example, paraquat exposure) and genetic factors have been investigated as putative causes of the disease. In the latter case, mutations or changes in the protein DJ-1 have been reported to be associated with autosomal recessive, early-onset parkinsonism. In this paper we established a model system to study the involvement of the DJ-1 protein in paraquat-induced autophagy. When human neuroblastoma SH-SY5Y cells were transfected with DJ-1-specific small interfering RNAs and exposed to paraquat, we observed (i) sensitization additive with paraquat-induced apoptotic cell death, (ii) inhibition of the cytoplasmic accumulation of autophagic vacuoles as well as the recruitment of LC3 fusion protein to the vacuoles, (iii) exacerbation of apoptotic cell death in the presence of the autophagy inhibitor 3-methyladenine, and (iv) an increase in mammalian target of rapamycin phosphorylation. Taken together, these findings suggest an active role for DJ-1 in the autophagic response produced by paraquat, providing evidence for the role of PD-related proteins in the autophagic degradation pathway, a factor that should be considered in the design of potential therapies for the treatment of the disease.     

Induction of autophagy promotes fusion of multivesicular bodies with autophagic vacuoles in k562 cells

Morphological and biochemical studies have shown that autophagosomes fuse with endosomes forming the so-called amphisomes, a prelysosomal hybrid organelle. In the present report, we have analyzed this process in K562 cells, an erythroleukemic cell line that generates multivesicular bodies (MVBs) and releases the internal vesicles known as exosomes into the extracellular medium. We have previously shown that in K562 cells, Rab11 decorates MVBs. Therefore, to study at the molecular level the interaction of MVBs with the autophagic pathway, we have examined by confocal microscopy the fate of MVBs in cells overexpressing green fluorescent protein (GFP)-Rab11 and the autophagosomal protein red fluorescent protein-light chain 3 (LC3). Autophagy inducers such as starvation or rapamycin caused an enlargement of the vacuoles decorated with GFP-Rab11 and a remarkable colocalization with LC3. This convergence was abrogated by a Rab11 dominant negative mutant, indicating that a functional Rab11 is involved in the interaction between MVBs and the autophagic pathway. Interestingly, we presented evidence that autophagy induction caused calcium accumulation in autophagic compartments. Furthermore, the convergence between the endosomal and the autophagic pathways was attenuated by the Ca2+ chelator acetoxymethyl ester (AM) of the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), indicating that fusion of MVBs with the autophagosome compartment is a calcium-dependent event. In addition, autophagy induction or overexpression of LC3 inhibited exosome release, suggesting that under conditions that stimulates autophagy, MVBs are directed to the autophagic pathway with consequent inhibition in exosome release.     

Identification of an autophagy defect in smokers' alveolar macrophages

Alveolar macrophages are essential for clearing bacteria from the alveolar surface and preventing microbe-induced infections. It is well documented that smokers have an increased incidence of infections, in particular lung infections. Alveolar macrophages accumulate in smokers' lungs, but they have a functional immune deficit. In this study, we identify an autophagy defect in smokers' alveolar macrophages. Smokers' alveolar macrophages accumulate both autophagosomes and p62, a marker of autophagic flux. The decrease in the process of autophagy leads to impaired protein aggregate clearance, dysfunctional mitochondria, and defective delivery of bacteria to lysosomes. This study identifies the autophagy pathway as a potential target for interventions designed to decrease infection rates in smokers and possibly in individuals with high environmental particulate exposure.     

The variability of autophagy and cell death susceptibility

Impaired autophagic machinery is implicated in a number of diseases such as heart disease, neurodegeneration and cancer. A common denominator in these pathologies is a dysregulation of autophagy that has been linked to a change in susceptibility to cell death. Although we have progressed in understanding the molecular machinery and regulation of the autophagic pathway, many unanswered questions remain. How does the metabolic contribution of autophagy connect with the cell’s history and how does its current autophagic flux affect metabolic status and susceptibility to undergo cell death? How does autophagic flux operate to switch metabolic direction and what are the underlying mechanisms in metabolite and energetic sensing, metabolite substrate provision and metabolic integration during the cellular stress response? In this article we focus on unresolved questions that address issues around the role of autophagy in sensing the energetic environment and its role in actively generating metabolite substrates. We attempt to provide answers by explaining how and when a change in autophagic pathway activity such as primary stress response is able to affect cell viability and when not. By addressing the dynamic metabolic relationship between autophagy, apoptosis and necrosis we provide a new perspective on the parameters that connect autophagic activity, severity of injury and cellular history in a logical manner. Last, by evaluating the cell’s condition and autophagic activity in a clear context of regulatory parameters in the intra- and extracellular environment, this review provides new concepts that set autophagy into an energetic feedback loop, that may assist in our understanding of autophagy in maintaining healthy cells or when it controls the threshold between cell death and cell survival.     

Highlights from this issue

Autophagy or Type II Programmed Cell Death (PCD) is a major intracellular pathway for the degradation and recycling of proteins, ribosomes and entire organelles. The role of this pathway in the anti-tumor effect of radiotherapy and in radiation toxicity is obscure. A complicated machinery of genes and proteins is involved in the regulation of autophagy as a response to a variety of stress factors including hypoxia, nutrient deprivation, cytotoxic agents and radiotherapy. Continuously accumulating data suggest that autophagic response of cancer cells to radiotherapy is a major pathway which, in contrast to apoptosis that leads to death, may lead to either death or cellular survival. A variety of agents have been recognized that induce or block autophagy, directly interfering with the cytotoxic effect of radiotherapy. Simultaneous targeting of autophagy and apoptosis during radiotherapy seems to further augment the anti-tumor effect. Radiobiology research should focus on the differential effect of fractionation on the induction of autophagy in different tumors and on the manipulation of this with autophagy triggering agents. Whether manipulation of this pathway in normal tissues may be used to confer cytoportection deserves also thorough investigation. Moreover, the role of pretreatment autophagic indices in tumor cells in predicting radiotherapy and chemotherapy outcome should be examined in translational studies.     

Dissecting the function of Atg1 complex in Dictyostelium autophagy reveals a connection with the pentose phosphate pathway enzyme transketolase

The network of protein–protein interactions of the Dictyostelium discoideum autophagy pathway was investigated by yeast two-hybrid screening of the conserved autophagic proteins Atg1 and Atg8. These analyses confirmed expected interactions described in other organisms and also identified novel interactors that highlight the complexity of autophagy regulation. The Atg1 kinase complex, an essential regulator of autophagy, was investigated in detail here. The composition of the Atg1 complex in D. discoideum is more similar to mammalian cells than to Saccharomyces cerevisiae as, besides Atg13, it contains Atg101, a protein not conserved in this yeast. We found that Atg101 interacts with Atg13 and genetic disruption of these proteins in Dictyostelium leads to an early block in autophagy, although the severity of the developmental phenotype and the degree of autophagic block is higher in Atg13-deficient cells. We have also identified a protein containing zinc-finger B-box and FNIP motifs that interacts with Atg101. Disruption of this protein increases autophagic flux, suggesting that it functions as a negative regulator of Atg101. We also describe the interaction of Atg1 kinase with the pentose phosphate pathway enzyme transketolase (TKT). We found changes in the activity of endogenous TKT activity in strains lacking or overexpressing Atg1, suggesting the presence of an unsuspected regulatory pathway between autophagy and the pentose phosphate pathway in Dictyostelium that seems to be conserved in mammalian cells.     

Carbon nanotubes,but not spherical nanoparticles,block autophagy by a shape-related targeting of lysosomes in murine macrophages

Nanoparticles (NPs) can be toxic, depending on their physico-chemical characteristics. Macroautophagy/autophagy could represent a potential underlying mechanism of this toxicity. We therefore set up a study aimed to characterize in depth the effects, on autophagy, of macrophage exposure to NPs, with a particular attention paid to the role of NP physico-chemical characteristics (specifically chemical composition, shape, size, length, crystal phase, and/or surface properties). We demonstrate that exposure to carbon nanotubes (CNT) but not to spherical NPs leads to the blockage of the autophagic flux. We further identified lysosomal dysfunction, in association with the downregulation of SNAPIN expression, as the underlying mechanism responsible for the CNT-induced autophagy blockade. These results identify for the first time the shape as a major determinant of the interaction of NPs with the autophagy pathway. Moreover, identifying the lysosomes and SNAPIN as primary targets of MWCNT toxicity opens new directions in the interpretation and understanding of nanomaterial toxicity.     

Clearance of dying autophagic cells of different origin by professional and non-professional phagocytes

MCF-7 cells undergo autophagic death upon tamoxifen treatment. Plated on non-adhesive substratum these cells died by anoikis while inducing autophagy as revealed by monodansylcadaverine staining, elevated light-chain-3 expression and electron microscopy. Both de novo and anoikis-derived autophagic dying cells were engulfed by human macrophages and MCF-7 cells. Inhibition of autophagy by 3-methyladenine abolished engulfment of cells dying through de novo autophagy, but not those dying through anoikis. Blocking exposure of phosphatidylserine (PS) on both dying cell types inhibited phagocytosis by MCF-7 but not by macrophages. Gene expression profiling showed that though both types of phagocytes expressed full repertoire of the PS recognition and signaling pathway, macrophages could evolve during engulfment of de novo autophagic cells the potential of calreticulin-mediated processes as well. Our data suggest that cells dying through autophagy and those committing anoikis with autophagy may engage in overlapping but distinct sets of clearance mechanisms in professional and non-professional phagocytes.     

NOD1 activates autophagy to aggravate hepatic ischemia-reperfusion injury in mice

Hepatic ischemia/reperfusion injury (IRI) is tissue damage resulting from return of the blood supply to the tissue after a period of ischemia or lack of oxygen. Much of the morbidity associated with liver transplantation and major hepatic resections is, in part, due to IRI. Both innate immunity and autophagy play important roles in hepatic IRI. With regard to innate immunity, one factor that plays a key role is NOD1, an intracellular pattern recognition receptor. NOD1 has recently been shown to be associated with autophagy, but the mechanisms involved with this process remain obscure. This relationship between NOD1 and autophagy prompted us to examine the role and potential mechanisms of NOD1 in regulating autophagy as related to hepatic IRI. We found that NOD1 was upregulated during hepatic IRI and was associated with an activation of the autophagic signaling pathway. Moreover, levels of Atg5, a critical protein associated with autophagy, were decreased when NOD1 was inhibited by NOD1 small interfering RNA. We conclude that NOD1 appears to exert a pivotal role in hepatic IRI by activating autophagy to aggravate hepatic IRI, and Atg5 was required for this process. The identification of this novel pathway, that links expression levels of NOD1 with Atg5-mediated autophagy, may provide new insights for the generation of novel protective therapies directed against hepatic IRI.     

Lysosomal basification and decreased autophagic flux in oxidatively stressed trabecular meshwork cells

Increasing evidence suggests oxidative damage as a key factor contributing to the failure of the conventional outflow pathway tissue to maintain appropriate levels of intraocular pressure, and thus increase the risk for developing glaucoma, a late-onset disease which is the second leading cause of permanent blindness worldwide. Autophagy is emerging as an essential cellular survival mechanism against a variety of stressors, including oxidative stress. Here, we have monitored, by using different methodologies (LC3-I to LC3-II turnover, tfLC3, and Cyto ID), the induction of autophagy and autophagy flux in TM cells subjected to a normobaric hyperoxic model of mild chronic oxidative stress. Our data indicate the MTOR-mediated activation of autophagy and nuclear translocation of TFEB in oxidatively stressed TM cells, as well as the role of autophagy in the occurrence of SA-GLB1/SA-β-gal. Concomitant with the activation of the autophagic pathway, TM cells grown under oxidative stress conditions displayed, however, reduced cathepsin (CTS) activities, reduced lysosomal acidification and impaired CTSB proteolytic maturation, resulting in decreased autophagic flux. We propose that diminished autophagic flux induced by oxidative stress might represent one of the factors leading to progressive failure of cellular TM function with age and contribute to the pathogenesis of primary open angle glaucoma.     

Neuronal macroautophagy: from development to degeneration

Macroautophagy, a lysosomal pathway responsible for the turnover of organelles and long-lived proteins, has been regarded mainly as an inducible process in neurons, which is mobilized in states of stress and injury. New studies show, however, that macroautophagy is also constitutively active in healthy neurons and is vital to cell survival. Neurons in the brain, unlike cells in the periphery, are protected from large-scale autophagy induction because they can use several different energy sources optimally, receive additional nutrients and neurotrophin support from glial cells, and benefit from hypothalamic regulation of peripheral nutrient supplies. Due to its exceptional efficiency, constitutive autophagy in healthy neurons proceeds in the absence of easily detectable autophagic vacuole intermediates. These intermediates can accumulate rapidly, however, when late steps in the autophagic process are blocked. Autophagic vacuoles also accumulate abnormally in affected neurons of several major neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease, where they have been linked to various aspects of disease pathogenesis including neuronal cell death. The build-up of autophagic vacuoles in these neurological disorders and others may reflect either heightened autophagy induction, impairment in later digestive steps in the autophagy pathway, or both. Determining the basis for AV accumulation is critical for understanding the pathogenic significance of autophagy in a given pathologic state and for designing possible therapies based on modulating autophagy. In this review, we discuss the special features of autophagy regulation in the brain, its suspected roles in neurodevelopment and plasticity, and recent progress toward understanding how dysfunctional autophagy contributes to neurodegenerative disease.