Downregulation Of Autophagy Research Articles

Atorvastatin Protects Vascular Smooth Muscle Cells From TGF-β1-Stimulated Calcification by Inducing Autophagy via Suppression of the β-Catenin Pathway

Background: Arterial calcification is a major event in the progression of atherosclerosis. It is reported that statins exhibit various protective effects against vascular smooth muscle cell (VSMC) inflammation and proliferation in cardiovascular remodeling. Although statins counteract atherosclerosis, the molecular mechanisms of statins on the calcium release from VSMCs have not been clearly elucidated. Methods: Calcium content of VSMCs was measured using enzyme-linked immunosorbent assay (ELISA). The expression of proteins involved in cellular transdifferentiation was analyzed by western blot. Cell autophagy was measured by fluorescence microscopic analysis for acridine orange staining and transmission electron microscopy analysis. The autophagic inhibitors (3-MA, chloroquine, NH₄Cl and bafilomycin A1) and β-catenin inhibitor JW74 were used to assess the effects of atorvastatin on autophagy and the involvement of β-catenin on cell calcification respectively. Furthermore, cell transfection was performed to overexpress β-catenin. Results: In VSMCs, atorvastatin significantly suppressed transforming growth factor-β1 (TGF-β1)-stimulated calcification, accompanied by the induction of autophagy. Downregulation of autophagy with autophagic inhibitors significantly suppressed the inhibitory effect of atorvastatin on cell calcification. Moreover, the beneficial effect of atorvastatin on calcification and autophagy was reversed by β-catenin overexpression. Conversely, JW74 supplement enhanced this effect. Conclusion: These data demonstrated that atorvastatin protect VSMC from TGF-β1-stimulated calcification by inducing autophagy through suppression of the β-catenin pathway, identifying autophagy induction might be a therapeutic strategy for use in vascular calcification.

VMP1 related autophagy and apoptosis in colorectal cancer cells: VMP1 regulates cell death

Vacuole membrane protein 1 (VMP1) is an autophagy-related protein and identified as a key regulator of autophagy in recent years. In pancreatic cell lines, VMP1-dependent autophagy has been linked to positive regulation of apoptosis. However, there are no published reports on the role of VMP1 in autophagy and apoptosis in colorectal cancers. Therefore, to address this gap of knowledge, we decided to interrogate regulation of autophagy and apoptosis by VMP1. We have studied the induction of autophagy by starvation and rapamycin treatment in colorectal cell lines using electron microscopy, immunofluorescence, and immunoblotting. We found that starvation-induced autophagy correlated with an increase in VMP1 expression, that VMP1 interacted with BECLIN1, and that siRNA mediated down-regulation of VMP1-reduced autophagy. Next, we examined the relationship between VMP1-dependent autophagy and apoptosis and found that VMP1 down-regulation sensitizes cells to apoptosis and that agents that induce apoptosis down-regulate VMP1. In conclusion, similar to its reported role in other cell types, VMP1 is an important regulator of autophagy in colorectal cell lines. However, in contrast to its role in pancreatic cell lines, in colorectal cancer cells, VMP1-dependent autophagy appears to be pro-survival rather than pro-cell death.

Combination of LC3 shRNA Plasmid Transfection and Genistein Treatment Inhibited Autophagy and Increased Apoptosis in Malignant Neuroblastoma in Cell Culture and Animal Models

Malignant neuroblastoma is an extracranial solid tumor that usually occurs in children. Autophagy, which is a survival mechanism in many solid tumors including malignant neuroblastoma, deters the efficacy of conventional chemotherapeutic agents. To mimic starvation, we used 200 nM rapamycin that induced autophagy in human malignant neuroblastoma SK-N-BE2 and IMR-32 cells in cell culture and animal models. Combination of microtubule associated protein light chain 3 short hairpin RNA (LC3 shRNA) plasmid transfection and genistein (GST) treatment was tested for inhibiting rapamycin-induced autophagy and promoting apoptosis. The best synergistic efficacy caused the highest decrease in cell viability due to combination of 50 nM LC3 shRNA plasmid transfection and 25 µM GST treatment in rapamycin-treated SK-N-BE2 cells while combination of 100 nM LC3 shRNA plasmid transfection and 25 µM GST treatment in rapamycin-treated IMR-32 cells. Quantitation of acidic vesicular organelles confirmed that combination of LC3 shRNA plasmid transfection and GST treatment prevented rapamycin-induced autophagy due to down regulation of autophagy promoting marker molecules (LC3 II, Beclin 1, TLR-4, and Myd88) and upregulation of autophagy inhibiting marker molecules (p62 and mTOR) in both cell lines. Apoptosis assays showed that combination therapy most effectively activated mitochondrial pathway of apoptosis in human malignant neuroblastoma in cell culture and animal models. Collectively, our current combination of LC3 shRNA plasmid transfection and GST treatment could serve as a promising therapeutic strategy for inhibiting autophagy and increasing apoptosis in human malignant neuroblastoma in cell culture and animal models.

Macronutrient Composition of the Diet Affects the Feeding-Mediated Down Regulation of Autophagy in Muscle of Rainbow Trout (O. mykiss)

Autophagy functions as an important catabolic mechanism by mediating the turnover of intracellular organelles and protein complexes through a lysosome dependent degradative pathway. Although the induction of autophagy by starvation has been extensively studied, we still know very little about how autophagy is regulated under normal nutritional conditions. The purpose of the present study was to characterize both in vivo and in vitro the response of the autophagy-lysosomal degradative pathway to nutrient (amino acids and carbohydrates) availability in the muscle of the carnivorous rainbow trout. We report that meal feeding is accompanied by a rapid activation of Akt, FoxO1 and the Target of Rapamycin (TOR) signaling pathways and a concomitant decrease of autophagosome formation. We also show that this effect occurs only when the proportion of dietary proteins increases at the expense of carbohydrates. Concurrently, our in vitro study on primary culture of trout muscle cells demonstrates an opposite effect of amino acids and glucose on the regulation of autophagy-lysosomal pathways. More specifically, the addition of amino acids in cell culture medium inhibited the formation of autophagosomes, whereas the addition of glucose had an opposite effect. The effect of amino acids was accompanied by an activation of TOR, considered as an important regulator of autophagosomal formation. However, the mechanisms involved in the effect of glucose were independent of Akt, TOR and AMPK and remain to be determined. Together, these results demonstrated the specific role of macronutrients as well as that of their interactions in the regulation of autophagy and highlight the interest to consider the macronutrient composition of the diets in the control of this degradative pathway.

Autophagy in the liver

Autophagy in the liver

Enhancing autophagy with activated protein C and rapamycin protects against sepsis-induced acute lung injury

Autophagy plays distinct roles in apoptosis and the inflammatory process. Understanding the role of autophagy in sepsis-induced acute lung injury (ALI) may provide new insights into developing novel therapeutic strategies for this group of patients. The aim of this study was to investigate the regulation of autophagy in the septic lung and to use pharmacologic agents to modulate autophagy to study its functional significance. Mice were subjected to cecal ligation and puncture (CLP) or a sham operation. At 1 hour after CLP, mice were treated with vehicle, activated protein C (APC), rapamycin, or bafilomycin A1. Mice were humanely killed at 4 or 24 hours after the operation or were observed for ≤ 7 days. CLP induced a systemic inflammatory response and significantly decreased survival. In lung tissue, increased leukocyte infiltration, inflammation, and apoptosis were observed. In contrast, autophagy was suppressed after CLP such that the expression of LC3II, Atg5, and Rab7 were downregulated. Rapamycin activated autophagy, limited the CLP-induced proinflammatory response, and downregulated apoptotic activity after CLP. The administration of APC after CLP had an effect similar to that of rapamycin. Both medications significantly improved survival 7 days after CLP. The downregulation of autophagy may lead to systemic inflammation and ALI after sepsis. The direct or indirect modification of autophagy using rapamycin or APC, respectively, resulted in improved survival. Enhancing or restoring autophagy early after sepsis seems to be a potential strategy for the treatment of sepsis-induced ALI.

Nandrolone attenuates aortic adaptation to exercise in rats

In this study, we investigated the interaction between exercise-induced mitochondrial adaptation of large vessels and the effects of chronic anabolic androgenic steroids (AASs). Four groups of Sprague-Dawley rats were studied: (i) sedentary, (ii) sedentary + nandrolone-treated, (iii) aerobic exercise trained, and (iv) trained + nandrolone-treated. Aerobic training increased the levels of aortic endothelial nitric oxide synthase (eNOS) and heme oxygenase-1 (HO-1) in accordance with improved acetylcholine-induced vascular relaxation. These beneficial effects were associated with induction of mitochondrial complexes I and V, increased mitochondrial DNA copy number, and greater expression of transcription factors involved in mitochondrial biogenesis/fusion. We also observed enhanced mitochondrial autophagy pathway activity, including increased conversion of LC3-I to LC3-II and greater expression of beclin1 and autophagy-related protein-7 (ATG7). The levels of thiobarbituric acid-reactive substances and protein carbonyls remained unchanged, whereas significant increases in catalase and mitochondrial manganese superoxide dismutase (MnSOD) levels were observed in the aortas of trained animals, when compared with sedentary controls. Nandrolone increased oxidative stress biomarkers and inhibited exercise-induced increases of eNOS, HO-1, catalase, and MnSOD expression. In addition, it also attenuated elevated peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and mitofusin-2 expression, and further up-regulated LC3II conversion, beclin1, ATG7, and dynamin-related protein-1 expression. These results demonstrate that nandrolone attenuates aortic adaptations to exercise by regulating mitochondrial dynamic remodelling, including down-regulation of mitochondrial biogenesis and intensive autophagy.

Abstract B97: The role of JNK isoforms in adenovirus-induced autophagy: Implications for cancer immunotherapy using oncolytic adenoviruses.

Abstract Glioblastoma multiforme is the most common and lethal form of primary brain tumor in adults. Targeted immunotherapy using recombinant oncolytic viruses is a promising strategy for the treatment of brain tumors. Delta-24-RGD is a highly selective and potent oncolytic adenovirus that induces autophagy in malignant gliomas. Data from our ongoing clinical trial suggested Delta-24-RGD initiated an anti-tumor immune response in patients. Importantly, autophagy promotes the presentation of viral antigens for activation of immune cells; therefore, we sought to elucidate the molecular mechanisms by which adenoviruses induce autophagy. Adenoviral replication induced phosphorylation of JNK that was required for autophagy. Our results indicated that JNK regulates autophagy through Bcl-2 phosphorylation and consequent dissociation of Bcl-2 and beclin 1. Contrary to starvation-induced autophagy, adenovirus-mediated autophagy was facilitated by both JNK1 and JNK2 isoforms of JNK. Moreover, our data indicated that adenoviral antigens were presented at the cell surface, which was ablated upon down-regulation of JNK or autophagy. We propose a JNK/autophagy-dependent mechanism for the immune presentation of viral antigens that can be leveraged when developing recombinant adenoviruses for the advancement of anti-tumor immunotherapy. Thus, we developed a genetically-modified Delta-24 adenovirus expressing an EGFRvIII epitope that is aberrantly expressed in gliomas. The majority (>80%) of cells infected with Delta-24-FvIII presented the fiber-bound EGFRvIII epitope on their surface, an effect that was diminished upon inhibition of autophagy. Our data demonstrated that oncolytic adenoviruses can be modified to present cancer antigens for an anti-tumor immune response that may be enhanced by positively regulating autophagy. Citation Format: Sarah R. Klein, Hong Jiang, Sujan Piya, Zhimin Lu, Candelaria Gomez-Manzano, Juan Fueyo. The role of JNK isoforms in adenovirus-induced autophagy: Implications for cancer immunotherapy using oncolytic adenoviruses. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B97.

Cyclin D1 Activity Regulates Autophagy and Senescence in the Mammary Epithelium

Overexpression of cyclin D1 is believed to endow mammary epithelial cells (MEC) with a proliferative advantage by virtue of its contribution to pRB inactivation. Accordingly, abrogation of the kinase-dependent function of cyclin D1 is sufficient to render mice resistant to breast cancer initiated by ErbB2. Here, we report that mouse cyclin D1(KE/KE) MECs (deficient in cyclin D1 activity) upregulate an autophagy-like process but fail to implement ErbB2-induced senescence in vivo. In addition, immortalized cyclin D1(KE/KE) MECs retain high rates of autophagy and reduced ErbB2-mediated transformation in vitro. However, highlighting its dual role during tumorigenesis, downregulation of autophagy led to an increase in senescence in cyclin D1(KE/KE) MECs. Autophagy upregulation was also confirmed in human mammary epithelial cells (HMEC) subjected to genetic and pharmacologic inhibition of cyclin D1 activity and, similar to our murine system, simultaneous inhibition of Cdk4/6 and autophagy in HMECs enhanced the senescence response. Collectively, our findings suggest a previously unrecognized function of cyclin D1 in suppressing autophagy in the mammary epithelium.

Abstract 19387: Targeted Deletion of Mmp9 Mitigates Autophagy Mediated Contractile Dysfunction in Insulin2 Mutant Diabetic Mice

Insulin mediated activation of MMP9 and myocardial autophagy fosters pathological cardiac remodeling, and targeted deletion of MMP9 improves contractility. We hypothesize that ablation of MMP9 mitigates autophagy mediated contractile dysfunction in diabetes. To test the hypothesis, we created Ins2+/- /MMP9-/- mice, where MMP9 gene is deleted from diabetic Ins2+/-. We measured the levels of LC3B-II and Atg3 (autophagy markers) in C57BL/6J, Ins2+/- and Ins2+/- /MMP9-/- hearts. The results revealed induction of both LC3B-II and Atg3 in Ins2+/-, which is mitigated in Ins2+/-/MMP9-/- (fold change in C57BL/6J, Ins2+/-, Ins2+/- /MMP9-/- for LC3B-II: 1; 4.5 ±0.3, 3.0± 0.2, and Atg3: 1, 2.5±0.2, 1±0.3, respectively). The down regulation of autophagy in Ins2+/-/MMP9-/- suggests that targeted deletion of MMP9 attenuates autophagy in diabetes. To assess the specific role of autophagy in contractility, we administered Mdivi-1 (autophagy blocker) into C57BL/6J (control) and Ins2+/- mice (50mg/Kg, i.p) for 7 days and determined the improvement in contractility (±dL/dt) of cardiomyocytes by Ion-Optics device. We found no significant difference in the rate of contraction (dL/dt) and relaxation (-dL/dt) in C57BL/6J untreated (dL/dt=160±13 and -dL/dt= -165± 15) and treated (dL/dt= 170±20 and -dL/dt= -165± 21) groups. However, there was significant improvement in ±dL/dt in Ins2+/- (untreated: dL/dt=100±10 and -dL/dt= -95± 12) and treated (dL/dt= 185± 17 and -dL/dt= -180± 14). These elicit a novel regulatory role of MMP9 in autophagy in diabetes. It also suggests that inhibition of autophagy improves contractility in diabetic hearts. In conclusion, targeted deletion of MMP9 mitigates autophagy mediated contractile dysfunction in diabetes.

Activation of serum/glucocorticoid‐induced kinase 1 (SGK1) is important to maintain skeletal muscle homeostasis and prevent atrophy

Maintaining skeletal muscle mass is essential for general health and prevention of disease progression in various neuromuscular conditions. Currently, no treatments are available to prevent progressive loss of muscle mass in any of these conditions. Hibernating mammals are protected from muscle atrophy despite prolonged periods of immobilization and starvation. Here, we describe a mechanism underlying muscle preservation and translate it to non-hibernating mammals. Although Akt has an established role in skeletal muscle homeostasis, we find that serum- and glucocorticoid-inducible kinase 1 (SGK1) regulates muscle mass maintenance via downregulation of proteolysis and autophagy as well as increased protein synthesis during hibernation. We demonstrate that SGK1 is critical for the maintenance of skeletal muscle homeostasis and function in non-hibernating mammals in normal and atrophic conditions such as starvation and immobilization. Our results identify a novel therapeutic target to combat loss of skeletal muscle mass associated with muscle degeneration and atrophy.

Targeting Androgen Receptor Leads to Suppression of Prostate Cancer via Induction of Autophagy

Androgen receptor has a critical role in prostate cancer development and progression. Cell death via autophagy may also contribute to prostate cancer progression. We determined the role and regulatory effects of androgen receptor on the autophagy process of prostate cancer cells. Using a series of morphological approaches, such as transmission electron microscopy, monodansylcadaverine (Sigma®) and GFP-LC3 fluorescence microscopy assay, and Western blot we monitored the autophagic process in 3 pairs of prostate cancer cell lines to study the relationship to androgen receptor signals. Androgen receptor knockdown in androgen receptor positive cells, such as LNCaP or CWRrv1 human prostate cancer cells, led to increased autophagy. Adding functional androgen receptor to androgen receptor negative cells, such as PC3 human prostate cancer cells, resulted in decreased autophagy. This suggests that androgen receptor could have a negative role in regulating autophagy. Mechanism dissection indicated that androgen receptor might repress autophagy via modulation of p62 expression. A therapeutic approach of targeting androgen receptor to increase autophagy using the androgen receptor degradation enhancer ASC-J9® suppressed prostate cancer growth. Findings provide evidence that the androgen receptor might promote prostate cancer cell growth via autophagy down-regulation. Targeting the androgen receptor via ASC-J9 might lead to tumor suppression via the induction of autophagy. This may represent a new, potential therapeutic approach and mechanism for prostate cancer.

PP2A blockade inhibits autophagy and causes intraneuronal accumulation of ubiquitinated proteins

Using cultured cortical neurons, we show that the blockade of protein phosphatase 2A (PP2A), either pharmacologically by okadaic acid or by short hairpin RNA (shRNA)-mediated silencing of PP2A catalytic subunit, inhibited basal autophagy and autophagy induced in several experimental settings (including serum deprivation, endoplasmic reticulum stress, rapamycin, and proteasome inhibition) at early stages before autophagosome maturation. Conversely, PP2A upregulation by PP2A catalytic subunit overexpression stimulates neuronal autophagy. In addition, PP2A blockade resulted in the activation of the negative regulator of autophagy mammalian target of rapamycin complex 1 and 5' adenosine monophosphate (AMP)-activated protein kinase (AMPK) and led to intraneuronal accumulation of p62- and ubiquitin-positive protein inclusions, likely due to autophagy downregulation. These data are consistent with previous findings showing that specific invalidation of the autophagy process in the nervous system of mouse resulted in the accumulation of p62- and ubiquitin-positive protein inclusion bodies. Furthermore, we showed that PP2A inhibition alters the distribution of the microtubule-associated protein 1 light chain(LC) 3-I (MAP LC3-I), a key component of the autophagy molecular machinery. Whether MAP LC3-I distribution in the cell accounts for autophagy regulation remains to be determined. These data are important to human neurodegenerative diseases, especially Alzheimer's disease, because they provide links for the first time between the pathological features of Alzheimer's disease:PP2A downregulation, autophagy disruption, and protein aggregation.

Autophagy and Apoptosis Act as Partners to Induce Germ Cell Death after Heat Stress in Mice

Testicular heating suppresses spermatogenesis which is marked by germ cell loss via apoptotic pathways. Recently, it is reported that autophagy also can be induced by heat treatment in somatic cells. In this study, the status of autophagy in germ cells after heat treatment, as well as the partnership between autophagy and apoptosis in these cells was investigated. The results demonstrated that besides initiating apoptotic pathways, heat also induced autophagic pathways in germ cells. Exposure of germ cells to hyperthermia resulted in several specific features of the autophagic process, including autophagosome formation and the conversion of LC3-I to LC3-II. Furthermore, the ubiquitin-like protein conjugation system was implicated as being likely responsible for heat-induced autophagy in germ cells since all genes involving this system were found to be expressed in the testes. In addition, the upstream protein in this system, Atg7 (Autophagy-related gene 7), was found to be expressed in all types of spermatogenic cells, and its expression level was positively correlated with the level of autophagy in germ cells. As a result, Atg7 was selected as the investigative target to further analyze the role of autophagy in heat-induced germ cell death. It was shown that down expression of Atg7 protein resulted in the notable decrease in the level of autophagy in heat-treated germ cells, and this down-regulation of autophagy caused by Atg7 knockdown further reduced the apoptotic rate of germ cells. These results suggest that autophagy plays a positive role in the process of germ cell apoptosis after heat treatment. In conclusion, this study demonstrates that heat triggers autophagy and apoptosis in germ cells. These two mechanisms might act as partners, not antagonist, to induce cell death and lead to eventual destruction of spermatogenesis.

Autophagy downregulates thrombin-induced VSMCs proliferation through lysosomal pathway

of ceroid which is an insoluble protein complex bound with oxidized lipids found in almost all atherosclerotic lesions [2]. Although there is evidence that induction of autophagy through beclin1 activation can inhibit cell proliferation in several cancer cell lines, while as yet, knowledge about the impact of autophagy in thrombin-induced VSMCs proliferation is still limited. Hence, we conducted the current research to uncover the link between autophagy and proliferation of VSMCs. The experimental VSMCs were obtained from the abdominal aorta of healthy SD rat and cultured. The passage 4 VSMCs were used in the experiments and were divided into the following groups: (1) control group, (2) thrombin group (1 U/ml), (3) thrombin (1 U/ ml)+trehalose group, (4) thrombin (1 U/ml)+3-methyladenine (3-MA) (10 ng/ml) group, (5) thrombin (1 U/ml)+3-methyladenine (3-MA)+pepstatin A group (10 ng/ml). Immunofluorescence and western blot analysis were used to detect the proliferation related protein PCNA expression after treatment of trehalose, which is a novel mTOR-independent autophagy enhancer, and autophagy inhibitor 3-methyladenine (3-MA). Thereafter, we further discussed the potential mechanism herein; pepstatin A was used to inhibit the lysosomal pathway to inspect its involvement in the process. Our results showed that autophagy up-regulation can suppress thrombin-induced proliferation of VSMCs, in contrast, down-regulation of autophagy played a positive role in thrombin-stimulated VSMCs proliferation, and furthermore, this function can be inhibited by the treatment of pepstatin A.Tofurtherunderstandthepotentialmechanismof theinhibitoryroleof autophagy in thrombin-induced VSMCs proliferation, the expression of PCNA was detected after inhibition of lysosomal signaling pathway with pepstatin A.Combination use of pepstatin Awith trehalosecan neutralize the down-regulated role of autophagy on thrombin-induced VSMCs proliferation, which suggested the involvement of autophagy–lysosome pathway in this mechanism. With the awareness of the essential significance of VSMCs proliferation in cardiovascular diseases, antiproliferative strategies have aroused tremendous attention. In the present stage, antiproliferative strategies mainly include long-term and shortterm treatments with therapeutic demerits both, for example, the shortterm cell-inhibitory agents may not be so effective for such a diffuse disease like atherosclerosis, while long-term agents may weaken the fibrous cap of lesions and followed by increased susceptibility to rupture [3,4]. Autophagy was initially described as a positive role for cell survival, defective autophagy is involved in various diseases such as infections, cardiovascular diseases and cancers, etc. [5], and it can be triggered by some factors such as oxidized lipids, inflammationstress states in terms of atherosclerosis [6]. In the current study, we have observed the expression of proliferating cell nuclear antigen (PCNA) and microtubule-associated protein light chain 3 (MAP1-LC3) under treatment of autophagy inducer trehalose and autophagy inhibitor 3methyladenine (3-MA) (Fig.1). We found that the thrombin-induced proliferation of VSMCs decreased under up-regulation of autophagy, while in the sharp contrast, down-regulation of autophagy can increase thrombin-stimulated VSMCs proliferation, which can be inhibited by the treatment of pepstatin A, suggesting the involvement of autophagy–lysosome signal pathway in this process. In this investigation, we chose trehalose as autophagy inducer rather than the classic one rapamycin, for the former one is an mTORindependent agent while the later increases cell proliferation through PI3K/mTOR signal pathway [7] .T hesefindings suggest that autophagy acts as an adverse effect in thrombin-induced VSMCs proliferation through autophagy–lysosome pathway. VSMCs antiproliferative strategies based upon the regulation of autophagy may emerge as a novel approach in atherosclerosis treatment. Several studies demonstratedthatectopicexpressionofautophagy-relatedgenebeclin1

Cytoprotective effects of hesperetin and hesperidin against amyloid β‐induced impairment of glucose transport through downregulation of neuronal autophagy

This study investigated whether flavonoids, such as hesperetin and hesperidin, inhibited amyloid β (Aβ)-impaired glucose utilization through regulating cellular autophagy in insulin-stimulated neuronal cells. In this study, we used a toxic Aβ1-42 peptide to impair insulin-stimulated glucose utilization in Neuro-2A cells, and this study also hypothesized that Aβ-induced autophagy might be emerging as a key process regulating neuronal glucose uptake. Additionally, hesperetin and hesperidin were used to test the neuroprotective effect against Aβ-induced impairment of glucose utilization. Our data found that Aβ-stimulated autophagy activation promoted the phenomenon of impairment of neuronal energy metabolism, including glucose uptake, glucose transporters (GLUTs), and insulin signaling cascades. In this study, confocal images of autophagy punctate further confirmed that downregulation of Aβ-stimulated autophagy could increase insulin-stimulated neuronal glucose uptake. Moreover, treatment with hesperetin and hesperidin improved Aβ-impaired glucose utilization by inhibiting Aβ-induced autophagy in neuronal cells. These findings suggest that downregulation of autophagy may be one of the approaches to control the impairment of energy metabolism leading to neuronal injury in the early development of Alzheimer's disease, and hesperetin or hesperidin may be a potential agent in the preventing of Alzheimer's disease progression.

Induced autophagy reduces virus output in dengue infected monocytic cells

While several studies have shown a role for autophagy in the replication of dengue virus (DENV), these studies have been performed in directly infected cells. However, in severe cases of DENV infection the critical cell in the disease is believed to be monocytes which are poorly infected directly, but are highly susceptible to antibody enhanced infection. This study sought to determine the involvement of autophagy in the DENV infection of monocytic cells, using U937 cells as a model system. While the induction of autophagy was seen in response to DENV-2 infection, biochemical induction of autophagy resulted in a significant decrease in virus output. Down regulation of autophagy resulted in only a very slight increase in intracellular virus levels. In monocytic cells autophagy is not a significant part of the DENV replication mechanism, and there are distinct cell type specific differences in the DENV–autophagy interaction.

Autophagy promotes survival of retinal ganglion cells after optic nerve axotomy in mice

Autophagy is an essential recycling pathway implicated in neurodegeneration either as a pro-survival or a pro-death mechanism. Its role after axonal injury is still uncertain. Axotomy of the optic nerve is a classical model of neurodegeneration. It induces retinal ganglion cell death, a process also occurring in glaucoma and other optic neuropathies. We analyzed autophagy induction and cell survival following optic nerve transection (ONT) in mice. Our results demonstrate activation of autophagy shortly after axotomy with autophagosome formation, upregulation of the autophagy regulator Atg5 and apoptotic death of 50% of the retinal ganglion cells (RGCs) after 5 days. Genetic downregulation of autophagy using knockout mice for Atg4B (another regulator of autophagy) or with specific deletion of Atg5 in retinal ganglion cells, using the Atg5(flox/flox) mice reduces cell survival after ONT, whereas pharmacological induction of autophagy in vivo increases the number of surviving cells. In conclusion, our data support that autophagy has a cytoprotective role in RGCs after traumatic injury and may provide a new therapeutic strategy to ameliorate retinal diseases.

High Fat Induces Altered Colonic Motility and Enteric Neuronal Apoptosis

Aims: Obesity has been associated with several gastrointestinal motility problems including constipation. Previous studies have demonstrated central neuronal loss related to high fat diet. To study the etiology of the obesity related gastrointestinal symptoms we investigated the effect of twelve weeks of high fat diet (HF, 23%) on C57/BL6 mice compared to regular diet (6% fat). In addition we assessed the in-vitro effects of fatty acids on enteric neurons. Materials and method: Eight week old C57/BL6 mice were treated for 12 weeks with HF diet and their body weight and stool pellet number/hour, stool wet weight, and dry weight and water content were recorded. Isotonic muscle recordings were performed on the proximal colons. In-vitro studies were performed on postnatal IM-PEN enteric neuronal cell line. The neuronal cells were exposed to different doses of palmitate for 24 hours and MTS assay (for cell viability) were performed. Cells were also assessed for apoptosis using western blot analysis and immunostaining for cleaved caspase-3. Autophagy was assessed by western blot analysis for LC-3 and Beclin. Results: At the end of twelve weeks mice fed with the HF diet had a significant weight gain compared to normal diet group (34.2 ± 2.3 % and 23.2 ± 2.5 %, P<0.05). Mice on HF diet had a significantly lower pellet frequency (P<0.01) and pellet water content, as well as wet and dry pellet weight, resembling a constipation phenotype (Figure 1). Colonic isotonic muscle recording showed significantly less EFS-induced contractions in HF diet fed mice compared to normal diet (P<0.05), indicating loss of cholinergic neuronal function. Enteric neurons treatedwith palmitate showed a dose dependent reduction in viability (Figure 2). Exposure of enteric neuronal cells to 0.5 and 1 mM palmitate resulted in a dose dependent increase in cleaved caspase-3, which was associated with an increase in expression of LC3B and Beclin (n=3 independent blots).In addition immunostaining for cleaved caspase-3 showed enhanced apoptosis in cells exposed to palmitate compared to vehicle (15.6 ± 1.8 % and 10.6 ± 1.4 %, respectively P<0.01). To determine the signal transduction pathway involving fatty acid induced enteric neuronal apoptosis we assessed the c-Jun, p-AKT and NF-κB pathways. Exposure to palmitate (0.5 mM) was associated with a significant decrease in c-Jun and NK-κB protein expression, while it had no effect on p-AKT expression. Conclusion: In summary our data demonstrates the effects of high fat diet on reducing colonic motility. Exposure to fatty acids induced a reduction in enteric neuronal viability and enhanced enteric neuronal apoptosis. This data suggests there is a role for fatty acid induced enteric neuronal apoptosis accompanied by activation of autophagy pathways and down-regulation of c-Jun and NK-κB in mediating high fat diet-induced colonic dysmotility.

Downregulation of autophagy by Bcl-2 promotes MCF7 breast cancer cell growth independent of its inhibition of apoptosis

The anti-apoptotic Bcl-2 protein, which confers oncogenic transformation and drug resistance in most human cancers, including breast cancer, has recently been shown to effectively counteract autophagy by directly targeting Beclin1, an essential autophagy mediator and tumor suppressor. However, it remains unknown whether autophagy inhibition contributes to Bcl-2-mediated oncogenesis. Here, by using a loss-of-function mutagenesis study, we show that Bcl-2-mediated antagonism of autophagy has a critical role in enhancing the tumorigenic properties of MCF7 breast cancer cells independent of its anti-apoptosis activity. A Bcl-2 mutant defective in apoptosis inhibition but competent for autophagy suppression promotes MCF7 breast cancer cell growth in vitro and in vivo as efficiently as wild-type Bcl-2. The growth-promoting activity of this Bcl-2 mutant is strongly correlated with its suppression of Beclin1-dependent autophagy, leading to sustained p62 expression and increased DNA damage in xenograft tumors, which may directly contribute to tumorigenesis. Thus, the anti-autophagic property of Bcl-2 is a key feature of Bcl-2-mediated oncogenesis and may in some contexts, serve as an attractive target for breast and other cancer therapies.