Autophagy is induced and modulated by cholesterol depletion through transcription of autophagy-related genes and attenuation of flux.

Perturbations to cellular homeostasis, including reduction of the cholesterol level, induce autophagy, a self-digestion process of cellular constituents through an autophagosomal-lysosomal pathway. In accord with its function as a membrane organizer and metabolic sentinel, the cellular response to cholesterol depletion comprises multiple phenomena, including the activation of transcriptional responses, accumulation of reactive oxygen species (ROS), and activation of stress-related signaling pathways. However, the molecular mechanisms by which cholesterol depletion regulates autophagy and the putative involvement of transcriptional responses, ROS and/or stress-related signaling in autophagy regulation in this biological context are not fully understood. Here, we find that cholesterol depletion regulates autophagy at three different levels. First, employing RNA-seq, we show that cholesterol depletion increases the expression of autophagy-related genes independent of ROS or JNK activity. Second, analysis of LC3 lipidation and intracellular localization, and of p62 levels and degradation kinetics, reveals that cholesterol depletion mediates autophagy induction while interfering with autophagic flux. Of note, only the latter depends on ROS accumulation and JNK activity. In view of the common use of cholesterol-reducing drugs as therapeutic agents, our findings have important implications for multiple cellular settings in which autophagy plays a prominent role.

Altered mitochondrial dynamics and function in APOE4-expressing astrocytes.

APOE4 is a major risk factor for sporadic Alzheimer's disease; however, it is unclear how it exerts its pathological effects. Others and we have previously shown that autophagy is impaired in APOE4 compared to APOE3 astrocytes, and demonstrated differences in the expression of mitochondrial dynamics proteins in brains of APOE3 and APOE4 transgenic mice. Here, we investigated the effect of APOE4 expression on several aspects of mitochondrial function and network dynamics, including fusion, fission, and mitophagy, specifically in astrocytes. We found that APOE3 and APOE4 astrocytes differ in their mitochondrial dynamics, suggesting that the mitochondria of APOE4 astrocytes exhibit reduced fission and mitophagy. APOE4 astrocytes also show impaired mitochondrial function. Importantly, the autophagy inducer rapamycin enhanced mitophagy and improved mitochondrial functioning in APOE4 astrocytes. Collectively, the results demonstrate that APOE4 expression is associated with altered mitochondrial dynamics, which might lead to impaired mitochondrial function in astrocytes. This, in turn, may contribute to the pathological effects of APOE4 in Alzheimer's disease.

Autophagy induction in the treatment of Alzheimer's disease.

A growing body of evidence indicates that autophagy, an intracellular degradation pathway, profoundly affects Alzheimer's disease (AD) pathogenesis. Autophagy mediates the degradation of neurotoxic material and damaged organelles, allowing their clearance by glial and neuronal cells, while impaired autophagy may account for the accumulation of protein aggregates. Accordingly, dysfunctional autophagy is one of AD hallmarks; it occurs early in the disease development, which makes it an attractive therapeutic intervention target. Therefore, in recent years, the potential of autophagy induction as a treatment for AD has been studied extensively using various autophagy inducers, most of which are already in clinical practice for other medical conditions. Albeit promising results, including in AD clinical trials, this therapeutic strategy still requires careful consideration in order to fully understand the role of autophagy in AD pathogenesis and to further improve the outcomes. This review summarizes the current findings in this field and raises open questions and new prospects.

The Effects of APOE4 on Mitochondrial Dynamics and Proteins in vivo.

This study examined the effects of apolipoprotein E4 (APOE4), the most prevalent genetic risk factor for Alzheimer's disease (AD), on proteins involved in mitochondrial dynamics and autophagy, in the hippocampus of targeted replacement mice. Immunohistochemical measurements revealed that the levels of the mitochondrial fusion-mediating protein, MFN1, were higher, whereas those of corresponding fission-regulating protein, DRP-1, were lower in the hippocampus of ApoE4 mice than in the corresponding ApoE3 mice, indicating that APOE4 is associated with increased mitochondrial fusion and decreased fission. A similar ApoE4-driven decrease in DRP-1 was also observed in AD brains. The levels of the mitochondrial proteins COX1 and Tom40, were higher in the ApoE4 mice, which is consistent with the increased fusion. Measurements of the levels of cleaved PINK1 and parkin, which mark and target mitochondria for mitophagic degradation, revealed lower levels of cleaved PINK1, suggesting reduced mitochondrial membrane potential, and higher levels of parkin in the hippocampus of ApoE4 compared with the ApoE3 mice, indicating altered mitophagy. The levels of the ubiquitin-binding scaffold protein, p62/SQSTM1, which directs selected cargo to the autophagosomes, were also higher in the ApoE4 mice. These findings suggest that APOE4 is associated with enhanced mitochondrial fusion and decreased fission. Additionally, the results indicate that mitophagy/autophagy is reduced in ApoE4 mice, resulting in higher levels of proteins such as parkin and p62, which are normally degraded during this process. Taken together, these results suggest a novel mechanism that may underlie the pathological effects of APOE4 and indicate that use of APOE4 genotyping could pave the way for identification of novel APOE4-related therapeutic targets.

Blood Glutamate Scavenger as a Novel Neuroprotective Treatment in Spinal Cord Injury.

Neurotrauma causes immediate elevation of extracellular glutamate (Glu) levels, which creates excitotoxicity and facilitates inflammation, glial scar formation, and consequently neuronal death. Finding factors that reduce the inflammatory response and glial scar formation, and increase neuronal survival and neurite outgrowth, are of major importance for improving the outcome after spinal cord injury (SCI). In the present study, we evaluated a new treatment aiming to remove central nervous system (CNS) Glu into the systemic blood circulation by intravenous (IV) administration of blood Glu scavengers (BGS) such as the enzyme recombinant glutamate-oxaloacetate transaminase 1 (rGOT1) and its co-substrate. In this study we induced in mice an SCI (hemisection), and 1 h post-injury started administering BGS treatment for 5 consecutive days. The treatment reduced the expression levels of p-p38, which regulates apoptosis and increased the expression of p-Akt, which mediates cell survival. Moreover, this treatment decreased pro-inflammatory cytokine expression and microglia activation, reduced astrocytes' reactivity, and facilitated expression of radial glia markers such as Pax6 and nestin. BGS treatment increased the survival of neurons at lesion site and enabled axonal regeneration into the injury site. These effects were correlated with improved functional recovery of the left paretic hindlimb. Thus, early pharmacological intervention with BGS following SCI may be neuroprotective and create a pro-regenerative environment by modulating glia cell response. In light of our results, the availability of the method to remove excess Glu from CNS without the need to deliver drugs across the blood-brain barrier (BBB) and with minimal or no adverse effects may provide a major therapeutic asset.

Nucleolin and ErbB2 inhibition reduces tumorigenicity of ErbB2-positive breast cancer.

ErbB2, a member of the ErbB family of receptor tyrosine kinases, is an essential player in the cell's growth and proliferation signaling pathways. Amplification or overexpression of ErbB2 is observed in ∼30% of breast cancer patients, and often drives cellular transformation and cancer development. Recently, we have shown that ErbB2 interacts with the nuclear-cytoplasmic shuttling protein nucleolin, an interaction which enhances cell transformation in vitro, and increases mortality risk and disease progression rate in human breast cancer patients. Given these results, and since acquired resistance to anti-ErbB2-targeted therapy is a major obstacle in treatment of breast cancer, we have examined the therapeutic potential of targeting the ErbB2-nucleolin complex. The effect of the nucleolin-specific inhibitor GroA (AS1411) on ErbB2-positive breast cancer was tested in vivo, in a mouse xenograft model for breast cancer; as well as in vitro, alone and in combination with the ErbB2 kinase-inhibitor tyrphostin AG-825. Here, we show that in vivo treatment of ErbB2-positive breast tumor xenografts with GroA reduces tumor size and leads to decreased ErbB2-mediated signaling. Moreover, we found that co-treatment of breast cancer cell lines with GroA and the ErbB2 kinase-inhibitor tyrphostin AG-825 enhances the anti-cancer effects exerted by GroA alone in terms of cell viability, mortality, migration, and invasiveness. We, therefore, suggest a novel therapeutic approach, consisting of combined inhibition of ErbB2 and nucleolin, which has the potential to improve breast cancer treatment efficacy.

DJ-1 deficiency impairs autophagy and reduces alpha-synuclein phagocytosis by microglia.

Parkinson's disease (PD) is a progressive neurodegenerative disorder, of which 1% of the hereditary cases are linked to mutations in DJ-1, an oxidative stress sensor. The pathological hallmark of PD is intercellular inclusions termed Lewy Bodies, composed mainly of α-Synuclein (α-Syn) protein. Recent findings have shown that α-Syn can be transmitted from cell to cell, suggesting an important role of microglia, as the main scavenger cells of the brain, in clearing α-Syn. We previously reported that the knock down (KD) of DJ-1 in microglia increased cells' neurotoxicity to dopaminergic neurons. Here, we discovered that α-Syn significantly induced elevated secretion of the proinflammatory cytokines IL-6 and IL-1ß and a significant dose-dependent elevation in the production of nitric oxide in DJ-1 KD microglia, compared to control microglia. We further investigated the ability of DJ-1 KD microglia to uptake and degrade soluble α-Syn, and discovered that DJ-1 KD reduces cell-surface lipid raft expression in microglia and impairs their ability to uptake soluble α-Syn. Autophagy is an important mechanism for degradation of intracellular proteins and organelles. We discovered that DJ-1 KD microglia exhibit an impaired autophagy-dependent degradation of p62 and LC3 proteins, and that manipulation of autophagy had less effect on α-Syn uptake and clearance in DJ-1 KD microglia, compared to control microglia. Further studies of the link between DJ-1, α-Syn uptake and autophagy may provide useful insights into the role of microglia in the etiology of the PD.

Continuous treatment with FTS confers resistance to apoptosis and affects autophagy.

High percentage of human cancers involves alteration or mutation in Ras proteins, including the most aggressive malignancies, such as lung, colon and pancreatic cancers. FTS (Salirasib) is a farnesylcysteine mimetic, which acts as a functional Ras inhibitor, and was shown to exert anti-tumorigenic effects in vitro and in vivo. Previously, we have demonstrated that short-term treatment with FTS also induces protective autophagy in several cancer cell lines. Drug resistance is frequently observed in cancer cells exposed to prolonged treatment, and is considered a major cause for therapy inefficiency. Therefore, in the present study, we examined the effect of a prolonged treatment with FTS on drug resistance of HCT-116 human colon cancer cells, and the involvement of autophagy in this process. We found that cells grown in the presence of FTS for 6 months have become resistant to FTS-induced cell growth inhibition and cell death. Furthermore, we discovered that the resistant cells exhibit altered autophagy, reduced apoptosis and changes in Ras-related signaling pathways following treatment with FTS. Moreover we found that while FTS induces an apoptosis-related cleavage of p62, the FTS-resistant cells were more resistant to apoptosis and p62 cleavage.

Impaired Autophagy in APOE4 Astrocytes.

Alzheimer's disease (AD) is the most prevalent form of dementia in elderly. Genetic studies revealed allelic segregation of the apolipoprotein E (ApoE) gene in sporadic AD and in families with higher risk of AD. The mechanisms underlying the pathological effects of ApoE4 are not yet entirely clear. Several studies indicate that autophagy, which plays an important role in degradation pathways of proteins, organelles and protein aggregates, may be impaired in AD. In the present study, we investigated the effects of ApoE4 versus the ApoE3 isoform on the process of autophagy in mouse-derived astrocytes. The results obtained reveal that under several autophagy-inducing conditions, astrocytes expressing ApoE4 exhibit lower autophagic flux compared to astrocytes expressing ApoE3. Using an in situ model, we examined the role of autophagy and the effects thereon of ApoE4 in the elimination of Aß plaques from isolated brain sections of transgenic 5xFAD mice. This revealed that ApoE4 astrocytes eliminate Aß plaques less effectively than the corresponding ApoE3 astrocytes. Additional experiments showed that the autophagy inducer, rapamycin, enhances Aß plaque degradation by ApoE4 astrocytes whereas the autophagy inhibitor, chloroquine, blocks Aß plaque degradation by ApoE3 astrocytes. Taken together, these findings show that ApoE4 impairs autophagy in astrocyte cultures and that this effect is associated with reduced capacity to clear Aß plaques. This suggests that impaired autophagy may play a role in mediating the pathological effects of ApoE4 in AD.

Enhancing FTS (Salirasib) efficiency via combinatorial treatment.

The Ras oncogene transmits signals, which regulate various cellular processes including cell motility, differentiation, growth and death. Since Ras signalling is abnormally activated in more than 30% of human cancers, Ras and its downstream signalling pathways are considered good targets for therapeutic interference. Ras is post-translationally modified by the addition of a farnesyl group, which permits its attachment to the plasma membrane. Exploiting this knowledge, a synthetic Ras inhibitor, S-trans, trans-farnesylthiosalicylic acid (FTS; Salirasib), was developed. FTS resembles the farnesylcysteine group of Ras, and acts as an effective Ras antagonist. In the present review, the effect of FTS in combination with various other drugs, as tested in vitro and in vivo, and its therapeutic potential are discussed. As reviewed, FTS cooperates with diverse therapeutic agents, which significantly improves treatment outcome. Therefore, combinations of FTS with other agents have a potential to serve as anti-cancer or anti-inflammatory therapies.

Ras and autophagy in cancer development and therapy.

Autophagy, a process of self-degradation and turnover of cellular components, plays a complex role in cancer. Evidence exists to show that autophagy may support tumor growth and cell survival, whereas it can also contribute to tumor suppression and have anti-survival characteristics in different cellular systems. Numerous studies have described the effects of various oncogenes and tumor suppressors on autophagy. The small GTPase Ras is an oncogene involved in the regulation of various cell-signaling pathways, and is mutated in 33% of human cancers. In the present review, we discuss the interplay between Ras and autophagy in relation to oncogenesis. It appears that Ras can upregulate or downregulate autophagy through several signaling pathways. In turn, autophagy can affect the tumorigenicity driven by Ras, resulting in either tumor progression or repression, depending on the cellular context. Furthermore, Ras inhibitors were shown to induce autophagy in several cancer cell lines.

Chloroquine synergizes with FTS to enhance cell growth inhibition and cell death.

The Ras family of small GTPases transmits extracellular signals that regulate cell growth, differentiation, motility and death. Ras signaling is constitutively active in a large number of human cancers. Ras can also regulate autophagy by affecting several signaling pathways including the mTOR pathway. Autophagy is a process that regulates the balance between protein synthesis and protein degradation. It is important for normal growth control, but may be defective in diseases. Previously, we have shown that Ras inhibition by FTS induces autophagy, which partially protects cancer cells and may limit the use of FTS as an anti-cancer drug. Since FTS is a non toxic drug we hypothesized that FTS and chloroquine (an autophagy inhibitor) will synergize in cell growth inhibition and cell death. Thus, in the present study, we explored the mechanism of each individual drug and their combined action. Our results demonstrate that in HCT-116 and in Panc-1 cells, FTS induces autophagy, which can be inhibited by chloroquine. Furthermore, the combined treatment synergistically decreased the number of viable cells. Interestingly, the combined treatment enhanced apoptotic cell death as indicated by increased sub-G1 cell population, increased Hoechst staining, activation of caspase 3, decrease in survivin expression and release of cytochrome c. Thus, chloroquine treatment may promote FTS-mediated inhibition of tumor cell growth and may stimulate apoptotic cell death.

Epizootic hemorrhagic disease virus induces and benefits from cell stress, autophagy, and apoptosis.

The mode and timing of virally induced cell death hold the potential of regulating viral yield, viral transmission, and the severity of virally induced disease. Orbiviruses such as the epizootic hemorrhagic disease virus (EHDV) are nonenveloped and cytolytic. To date, the death of cells infected with EHDV, the signal transduction pathways involved in this process, and the consequence of their inhibition have yet to be characterized. Here, we report that the Ibaraki strain of EHDV2 (EHDV2-IBA) induces apoptosis, autophagy, a decrease in cellular protein synthesis, the activation of c-Jun N-terminal kinase (JNK), and the phosphorylation of the JNK substrate c-Jun. The production of infectious virions decreased upon inhibition of apoptosis with the pan-caspase inhibitor Q-VD-OPH (quinolyl-valyl-O-methylaspartyl-[-2,6-difluorophenoxy]-methyl ketone), upon inhibition of autophagy with 3-methyladenine or via the knockout of the autophagy regulator Atg5, or upon treatment of infected cells with the JNK inhibitor SP600125 or the cyclin-dependent kinase (CDK) inhibitor roscovitine, which also inhibited c-Jun phosphorylation. Moreover, Q-VD-OPH, SP600125, and roscovitine partially reduced EHDV2-IBA-induced cell death, and roscovitine diminished the induction of autophagy by EHDV2-IBA. Taken together, our results imply that EHDV induces and benefits from the activation of signaling pathways involved in cell stress and death.

Ras inhibition enhances autophagy, which partially protects cells from death.

Autophagy, a process of regulated turnover of cellular constituents, is essential for normal growth control but may be defective under pathological conditions. The Ras/PI3K/mTOR signaling pathway negatively regulates autophagy. Ras signaling has been documented in a large number of human cancers. In this in-vitro study we examined the effect of the Ras inhibitor Salirasib (S-trans, trans-farnesylthiosalicylic acid; FTS) on autophagy induction and cell viability. We show that Ras inhibition by FTS induced autophagy in several cell lines, including mouse embryonic fibroblasts and the human cancer cell lines HeLa, HCT-116 and DLD-1. The autophagy induced by FTS seems to inhibit the cell death induced by FTS, since in the absence of autophagy the death of FTS-treated cells was enhanced. Therefore, inhibition of autophagy may promote the inhibition of tumor cell growth and the cell death mediated by FTS.

Neuregulin promotes incomplete autophagy of prostate cancer cells that is independent of mTOR pathway inhibition.

BACKGROUND: Growth factors activating the ErbB receptors have been described in prostate tumors. The androgen dependent prostate cancer cell line, LNCaP, expresses the ErbB-1, ErbB-2 and ErbB-3 receptor tyrosine kinases. Previously, it was demonstrated that NRG activates ErbB-2/ErbB-3 heterodimers to induce LNCaP cell death, whereas, EGF activates ErbB-1/ErbB-1 or ErbB-1/ErbB-2 dimers to induce cell growth and survival. It was also demonstrated that PI3K inhibitors repressed this cell death suggesting that in androgen deprived LNCaP cells, NRG activates a PI3K-dependent pathway associated with cell death. METHODOLOGY/PRINCIPAL FINDINGS: In the present study we demonstrate that NRG induces autophagy in LNCaP cells, using LC3 as a marker. However, the autophagy induced by NRG may be incomplete since p62 levels elevate. We also demonstrated that NRG- induced autophagy is independent of mammalian target of rapamycin (mTOR) inhibition since NRG induces Akt and S6K activation. Interestingly, inhibition of reactive oxygen species (ROS) by N-acetylcysteine (NAC), inhibited NRG-induced autophagy and cell death. Our study also identified JNK and Beclin 1 as important components in NRG-induced autophagy and cell death. NRG induced elevation in JNK phosphorylation that was inhibited by NAC. Moreover, inhibitor of JNK inhibited NRG-induced autophagy and cell death. Also, in cells overexpressing Bcl-2 or cells expressing sh-RNA against Beclin 1, the effects of NRG, namely induction of autophagy and cell death, were inhibited. CONCLUSIONS/SIGNIFICANCE: Thus, in LNCaP cells, NRG-induces incomplete autophagy and cell death that depend on ROS levels. These effects of NRG are mediated by signaling pathway that activates JNK and Beclin 1, but is independent of mTOR inhibition.

Beclin 1 self-association is independent of autophagy induction by amino acid deprivation and rapamycin treatment.

Autophagy, a process of self-digestion of cellular constituents, regulates the balance between protein synthesis and protein degradation. Beclin 1 represents an important component of the autophagic machinery. It interacts with proteins that positively regulate autophagy, such as Vps34, UVRAG, and Ambra1, as well as with anti-apoptotic proteins such as Bcl-2 via its BH3-like domain to negatively regulate autophagy. Thus, Beclin 1 interactions with several proteins may regulate autophagy. To identify novel Beclin 1 interacting proteins, we utilized a GST-Beclin 1 fusion protein. Using mass spectroscopic analysis, we identified Beclin 1 as a protein that interacts with GST-Beclin 1. Further examination by cross linking and co-immunoprecipitation experiments confirmed that Beclin 1 self-interacts and that the coiled coil and the N-terminal region of Beclin 1 contribute to its oligomerization. Importantly, overexpression of vps34, UVRAG, or Bcl-x(L), had no effect on Beclin 1 self-interaction. Moreover, this self-interaction was independent of autophagy induction by amino acid deprivation or rapamycin treatment. These results suggest that full-length Beclin 1 is a stable oligomer under various conditions. Such an oligomer may provide a platform for further protein-protein interactions.