Selenium Deficiency Induces Autophagy in Immune Organs of Chickens

The aim of the present study was to investigate the effects of selenium (Se) deficiency on autophagy-related genes and on ultrastructural changes in the spleen, bursa of Fabricius, and thymus of chickens. The Se deficiency group was fed a basal diet containing Se at 0.033 mg/kg and the control group was fed the same basal diet containing Se at 0.15 mg/kg. The messenger RNA (mRNA) levels of the autophagy genes microtubule-associated protein 1 light chain 3 (LC3)-I, LC3-II, Beclin 1, dynein, autophagy associated gene 5 (ATG5), and target of rapamycin complex 1 (TORC1) were assessed using real-time qPCR. The protein levels of LC3-II, Beclin 1, and dynein were investigated using western blot analysis. Furthermore, the ultrastructure was observed using an electron microscope. The results indicated that spleen mRNA levels of LC3-I, LC3-II, Beclin 1, dynein, ATG5, and TORC1 and the protein levels of LC3-II, Beclin 1, and dynein were increased in the Se deficiency group compared with the control group. In the bursa of Fabricius, the mRNA levels of LC3-I, LC3-II, Beclin 1, dynein, ATG5, and TORC1 and the protein levels of Beclin 1 and dynein were increased; furthermore, the protein level of LC3-II was decreased in the Se deficiency group compared to the control group. In the thymus, the mRNA levels of LC3-I, Beclin 1, and ATG5 increased; the levels of LC3-II, dynein, and TORC1 were decreased; the protein level of Beclin 1 increased; and the levels of LC3-II and dynein decreased in the Se deficiency group compared to those in the control group. Further cellular morphological changes, such as autophagy vacuoles, autolysosomes, and lysosomal degradation, were observed in the spleen, bursa of Fabricius, and thymus of the Se-deficiency group. In summary, Se deficiency caused changes in autophagy-related genes, which increased the autophagic process and also caused structural damages to the immune organs of chickens.     

BCL2L11/BIM

In response to toxic stimuli, BCL2L11 (also known as BIM), a BH3-only protein, is released from its interaction with dynein light chain 1 (DYNLL1 also known as LC8) and can induce apoptosis by inactivating anti-apoptotic BCL2 proteins and by activating BAX-BAK1. Recently, we discovered that BCL2L11 interacts with BECN1 (Beclin 1), and that this interaction is facilitated by DYNLL1. BCL2L11 recruits BECN1 to microtubules by bridging BECN1 and DYNLL1, thereby inhibiting autophagy. In starvation conditions, BCL2L11 is phosphorylated by MAPK8/JNK and this phosphorylation abolishes the BCL2L11-DYNLL1 interaction, allowing dissociation of BCL2L11 and BECN1, thereby ameliorating autophagy inhibition. This finding demonstrates a novel function of BIM beyond its roles in apoptosis, highlighting the crosstalk between autophagy and apoptosis, and suggests that BCL2L11’s dual effects in inhibiting autophagy and promoting apoptosis may have important roles in disease pathogenesis.     

The proapoptotic activity of the Bcl-2 family member Bim is regulated by interaction with the dynein motor complex

Bcl-2 family members that have only a single Bcl-2 homology domain, BH3, are potent inducers of apoptosis, and some appear to play a critical role in developmentally programmed cell death. We examined the regulation of the proapoptotic activity of the BH3-only protein Bim. In healthy cells, most Bim molecules were bound to LC8 cytoplasmic dynein light chain and thereby sequestered to the microtubule-associated dynein motor complex. Certain apoptotic stimuli disrupted the interaction between LC8 and the dynein motor complex. This freed Bim to translocate together with LC8 to Bcl-2 and to neutralize its antiapoptotic activity. This process did not require caspase activity and therefore constitutes an initiating event in apoptosis signaling.     

Unleashing the Ambra1-Beclin 1 complex from dynein chains: Ulk1 sets Ambra1 free to induce autophagy

The Beclin 1-VPS34 complex plays a crucial role in the induction of the autophagic process by generating PtdIns(3)P-rich membranes, which act as platforms for ATG protein recruitment and autophagosome nucleation. Several cofactors, such as Ambra1, ATG14 and UVRAG, are necessary for Beclin 1 complex activity. However, the mechanism by which Beclin 1 complex activity is: stimulated by autophagic stimuli has not yet been fully elucidated. Recently, we reported that autophagosome formation in mammalian cells is primed by Ambra1 release from the dynein motor complex. We found that Ambra1 specifically binds the dynein motor complex under normal conditions through a direct interaction with DLC1. When autophagy is induced, Ambra1-DLC1 are released from the dynein complex in an ULK1-dependent manner, and relocalize to the endoplasmic reticulum, thus enabling autophagosome nucleation. In addition, we found that both DLC1 downregulation and Ambra1 mutations in its DLC1-binding sites strongly enhance autophagosome formation. Ambra1 is therefore not only a cofactor of Beclin 1 in favoring its kinase-associated activity, but also a crucial upstream regulator of autophagy initiation.     

Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy

Apoptosis and autophagy are both tightly regulated biological processes that play a central role in tissue homeostasis, development, and disease. The anti-apoptotic protein, Bcl-2, interacts with the evolutionarily conserved autophagy protein, Beclin 1. However, little is known about the functional significance of this interaction. Here, we show that wild-type Bcl-2 antiapoptotic proteins, but not Beclin 1 binding defective mutants of Bcl-2, inhibit Beclin 1-dependent autophagy in yeast and mammalian cells and that cardiac Bcl-2 transgenic expression inhibits autophagy in mouse heart muscle. Furthermore, Beclin 1 mutants that cannot bind to Bcl-2 induce more autophagy than wild-type Beclin 1 and, unlike wild-type Beclin 1, promote cell death. Thus, Bcl-2 not only functions as an antiapoptotic protein, but also as an antiautophagy protein via its inhibitory interaction with Beclin 1. This antiautophagy function of Bcl-2 may help maintain autophagy at levels that are compatible with cell survival, rather than cell death.     

Structure and dynamics of LC8 complexes with KXTQT-motif peptides: swallow and dynein intermediate chain compete for a common site

The dynein light chain LC8 is an integral subunit of the cytoplasmic dynein motor complex that binds directly to and promotes assembly of the dynein intermediate chain (IC). LC8 interacts also with a variety of putative dynein cargo molecules such as Bim, a proapoptotic Bcl2 family protein, which have the KXTQT recognition sequence and neuronal nitric oxide synthase (nNOS), which has the GIQVD fingerprint but shares the same binding grooves at the LC8 dimer interface. The work reported here investigates the interaction of LC8 with IC and a putative cargo, Swallow, which share the KXTQT recognition sequence, and addresses the apparent paradox of how LC8, as part of dynein, mediates binding to cargo. The structures of Drosophila LC8 bound to peptides from IC and Swallow solved by X-ray diffraction show that the IC and Swallow peptides bind in the same grooves at the dimer interface. Differences in flexibility between bound and free LC8 were evaluated from hydrogen isotope exchange experiments using heteronuclear NMR spectroscopy. Peptide binding causes an increase in protection from exchange primarily in residues that interact directly with the peptide, such as the beta-strand intertwined at the interface and the N-terminal end of helix alpha2. There is considerably more protection upon Swallow binding, consistent with tighter binding relative to IC. Comparison with the LC8/nNOS complex shows how both the GIQVD and KXTQT fingerprints are recognized in the same groove. The similar structures of LC8/IC and LC8/Swa and the tighter binding of Swallow call into question the role for LC8 as a cargo adaptor protein, and suggest that binding of LC8 to Swallow serves another function, possibly that of a dimerization engine, which is independent of its role in dynein.     

Autophagy promotes T-cell survival through degradation of proteins of the cell death machinery

Autophagy is implicated in regulating cell death in activated T cells, but the underlying mechanism is unclear. Here, we show that inhibition of autophagy via Beclin 1 gene deletion in T cells leads to rampant apoptosis in these cells upon TCR stimulation. Beclin 1-deficient mice fail to mount autoreactive T-cell responses and are resistant to experimental autoimmune encephalomyelitis. Compared with Th17 cells, Th1 cells are much more susceptible to cell death upon Beclin 1 deletion. Cell death proteins are highly increased in Beclin 1-deficient T cells and inhibition of caspases and genetic deletion of Bim reverse apoptosis. In addition, p62/sequestosome 1 binds to caspase-8 but does not control levels of procaspase-8 or other cell death-related proteins. These results establish a direct role of autophagy in inhibiting the programmed cell death through degradation of apoptosis proteins in activated T cells.     

Dapper1 promotes autophagy by enhancing the Beclin1-Vps34-Atg14L complex formation

Autophagy is an intracellular degradation process to clear up aggregated proteins or aged and damaged organelles. The Beclin1-Vps34-Atg14L complex is essential for autophagosome formation. However, how the complex formation is regulated is unclear. Here, we show that Dapper1 (Dpr1) acts as a critical regulator of the Beclin1-Vps34-Atg14L complex to promote autophagy. Dpr1 ablation in the central nervous system results in motor coordination defect and accumulation of p62 and ubiquitinated proteins. Dpr1 increases autophagosome formation as indicated by elevated puncta formation of LC3, Atg14L and DFCP1 (Double FYVE-containing protein 1). Conversely, loss of Dpr1 impairs LC3 lipidation and causes p62/SQSTM1 accumulation. Dpr1 directly interacts with Beclin1 and Atg14L and enhances the Beclin1-Vps34 interaction and Vps34 activity. Together, our findings suggest that Dpr1 enhances the Atg14L-Beclin1-Vps34 complex formation to drive autophagy.     

Autophagy: An overlooked mechanism of HIV-1 pathogenesis and NeuroAIDS?

Human immunodeficiency virus type 1 (HIV-1) establishes a persistent infection characterized by progressive depletion of CD4⁺ lymphocytes and immunosuppression. Although extensive research has examined the importance of apoptosis as a cause of cell death associated with HIV-1 infection, the role of autophagy has been largely ignored. Our laboratory has examined the autophagic process in HIV-1-infected cells. Following infection of human peripheral blood CD4+ T-cells or U937 cells with HIV-1 for 48 hours, the autophagy proteins Beclin 1 and LC3-II were found to be markedly decreased. Beclin 1 mRNA expression and autophagosomes were also reduced in HIV-1 infected cells. Thus, our data indicate that HIV-1 infection inhibits autophagy in infected cells in contrast to the previously described induction of autophagy by gp120 in uninfected bystander cells. It is likely that HIV-1 has evolved this mechanism as part of an elaborate attempt to evade the immune system while promoting its own replication. We believe that autophagy is an overlooked mechanism in HIV-1 pathogenesis and plays a particularly important role in the early cognitive impairment and dementia often associated with advanced AIDS. A model is presented that describes the potential role of autophagy in NeuroAIDS.

Addendum to: Zhou D, Spector SA. Human immunodeficiency virus type-1 infection inhibits autophagy. Aids 2008;22:695-9.     

Ambra1 modulates starvation-induced autophagy through AMPK signaling pathway in cardiomyocytes

Recent research has revealed a role for Ambra1, an autophagy-related gene-related (ATG) protein, in the autophagic pro-survival response, and Ambra1 has been shown to regulate Beclin1 and Beclin1-dependent autophagy in embryonic stem cells and cancer cells. However, whether Ambra1 plays an important role in the autophagy pathway in cardiomyocytes is unknown. In this study, we hypothesized that Ambra1 is an important regulator of autophagy and apoptosis in cardiomyocytes. To test this hypothesis, we confirmed autophagic activity in serum-starved cardiomyocytes by assessing endogenous microtubule-associated protein 1 light chain 3 (LC3) localization, the presence of autophagosomes and LC3 protein levels. Cell apoptosis and viability were measured by annexin-V and PI staining and MTT assays. We determined that serum deprivation-induced autophagy was associated with Ambra1 upregulation in cardiomyocytes. When Ambra1 expression was reduced by siRNA, the cardiomyocytes were more sensitive to staurosporine-induced apoptosis. In addition, co-immunoprecipitation of Ambra1 and Beclin1 demonstrated that Ambra1 and Beclin1 interact in serum-starved or rapamycin-treated cardiomyocytes, suggesting that Ambra1 regulates autophagy in cardiomyocytes by interacting with Beclin1. Finally, we determined that starvation stress-induced activation of Ambra1 contributes to the attenuation of adaptive AMP-activated protein kinase (AMPK) signaling. In conclusion, Ambra1 is a crucial regulator of autophagy and apoptosis through AMPK signaling pathway in cardiomyocytes that maintains the balance between autophagy and apoptosis.     

The Bcl-2 Homology Domain 3 Mimetic Gossypol Induces Both Beclin 1-dependent and Beclin 1-independent Cytoprotective Autophagy in Cancer Cells

Gossypol, a natural Bcl-2 homology domain 3 mimetic compound isolated from cottonseeds, is currently being evaluated in clinical trials. Here, we provide evidence that gossypol induces autophagy followed by apoptotic cell death in both the MCF-7 human breast adenocarcinoma and HeLa cell lines. We first show that knockdown of the Bcl-2 homology domain 3-only protein Beclin 1 reduces gossypol-induced autophagy in MCF-7 cells, but not in HeLa cells. Gossypol inhibits the interaction between Beclin 1 and Bcl-2 (B-cell leukemia/lymphoma 2), antagonizes the inhibition of autophagy by Bcl-2, and hence stimulates autophagy. We then show that knockdown of Vps34 reduces gossypol-induced autophagy in both cell lines, and consistent with this, the phosphatidylinositol 3-phosphate-binding protein WIPI-1 is recruited to autophagosomal membranes. Further, Atg5 knockdown also reduces gossypol-mediated autophagy. We conclude that gossypol induces autophagy in both a canonical and a noncanonical manner. Notably, we found that gossypol-mediated apoptotic cell death was potentiated by treatment with the autophagy inhibitor wortmannin or with small interfering RNA against essential autophagy genes (Vps34, Beclin 1, and Atg5). Our findings support the notion that gossypol-induced autophagy is cytoprotective and not part of the cell death process induced by this compound.     

Acute Phase Expression Pattern of ZnTs,LC3, and Beclin-1 in Rat Hippocampus and Its Regulation by 3-Methyladenine Following Recurrent Neonatal Seizures

It has been reported that autophagy and zinc transporters (ZnTs) both play the key roles in excitotoxicity, which is associated with cognitive deficits following developmental seizures. However, the influence of autophagy on acute phase ZnTs expression has never been studied. The present study sought to investigate the contribution of an autophagy inhibitor (3-methyladenine, 3-MA) on the regulation of ZnTs, microtubule-associated protein 1A/1B light chain 3 (LC3), and beclin-1 expression in rat hippocampus following recurrent neonatal seizures. We examined the expression of ZnT1∼ZnT3, LC3, and beclin-1 at 1.5, 3, 6, and 24 h after the last seizures using real-time RT-PCR and Western blot methods, respectively. The results showed that there were upregulated expressions of ZnT-1, ZnT-2, LC3, and beclin-1 of RS group. Pretreatment with 3-MA remarkably attenuated seizure-induced ZnT-1, ZnT-2, LC3, and beclin-1 increase. Additionally, linear correlations could be observed between LC3–Beclin1, LC3–ZnT-2, Beclin1–ZnT2, Beclin1–ZnT3, and among ZnT1∼ZnT3 in control group, while the linear correlations could be observed between LC3–Beclin1, Beclin1–ZnT2, and Beclin1–ZnT3 in RS group. These results demonstrate, for the first time, that there exists an interaction of Zn²⁺ with autophagic signals that are immediately activated in hippocampus after recurrent neonatal seizures, which might play a key role in neonatal seizure-induced excitotoxicity.     

Plac8‐mediated autophagy regulates nasopharyngeal carcinoma cell function via AKT/mTOR pathway

To explore the relationship between autophagy and cell function, we investigated how PLAC8‐mediated autophagy influences proliferation, apoptosis and epithelial‐mesenchymal transition (EMT) in NPC. Colony formation analyses and CCK8 assays were used to assess the proliferative capacity of NPC cells. Transmission electron microscopy (TEM) was used to identify autophagosomes. Autophagic flux was monitored using the tandem monomeric RFP‐GFP‐tagged LC3 (tfLC3) assay. The rate of apoptosis in NPC cells was analysed by flow cytometry. Western blot analysis was used to evaluate the activation of autophagy and the signalling status of the AKT/mTOR pathway. Our study reveals that knocking out PLAC8 (koPLAC8) induces autophagy and apoptosis, while suppressing NPC cell proliferation and EMT. However, inhibition of autophagy with 3‐methyladenine or by knocking down Beclin‐1 reverses the cell proliferation, apoptosis and EMT influenced by koPLAC8. We find that koPLAC8 inhibits the phosphorylation of AKT and its downstream target, mTOR. Moreover, immunofluorescence and co‐immunoprecipitation reveal complete PLAC8/AKT colocalization and PLAC8/AKT interaction, respectively. Furthermore, knockout of PLAC8 induced autophagy and inactivated AKT/mTOR signalling pathway of NPC xenografts. Overall, our findings demonstrate that koPLAC8 induces autophagy via the AKT/mTOR pathway, thereby inhibiting cell proliferation and EMT, and promoting apoptosis in NPC cells.     

Regulation of Beclin 1 in autophagy

Class III phosphatidylinositol 3-kinase (PI3KC3) plays a pleiotropic role in autophagy and protein sorting pathways. The human core complex of PI3KC3 consists of three major components including PI3KC3/hVps34, p150 and Beclin 1. How the specificity of PI3KC3 complex is derived towards autophagy is not clear. Utilizing a sequential affinity purification coupled with Mass spectrometry approach, we have successfully purified a human Beclin 1 complex and cloned a novel protein we called Barkor (Beclin 1-associated autophagy-related key regulator). The function of Barkor in autophagy has been manifested in several assays, including stress-induced LC3 lipidation, autophagosome formation, and Salmonella typhimurium amplification. Mechanistically, Barkor competes with UV radiation resistance associated gene product (UVRAG) for interaction with Beclin 1, and orients Beclin1 to autophagosomes. Barkor shares considerable sequence homology with Atg14 in yeast, representing an evolutionary conserved autophagy specific regulatory step in early autophagosome formation.     

DJ‐1 inhibits autophagy activity of prostate cancer cells by repressing JNK–Bcl2–Beclin1 signaling

The regulation of DJ‐1 on AR signaling plays an important role in the pathogenesis of prostate cancer (PCa). DJ‐1 could alter autophagy and regulate Beclin1‐involved autophagy response through JNK‐dependent pathway. JNK is known to mediate autophagy through Bcl2–Beclin1 complex. Therefore, this study aimed to investigate the significance of autophagy in DJ‐1‐modulated PCa cells. The current studies showed that DJ‐1 overexpression in LNCaP decreased LC3 transformation and autophagosome formation. However, DJ‐1 knockdown exerted the opposite effect. Moreover, DJ‐1 silencing inhibited survival and promoted death in LNCaP, which was recovered by autophagy inhibition with 3‐MA. In addition, DJ‐1 overexpression inhibited the phosphorylation of JNK and Bcl2, and the dissociation of Beclin1 and Bcl2; while the effect of silencing DJ‐1 was completely opposite. More important, JNK activated by anisimycin inhibited the proliferation and promoted death of DJ‐1‐overexpressed LNCaP while increasing LC3 transformation and LC3‐puncta formation, but these results were reversed by the decrease of Beclin1 (by spautin‐1). In contrast, when DJ‐1 was silenced, the death of LNCaP, LC3 transformation, and LC3‐puncta formation were inhibited by JNK inhibitor SP600125, which promoted cell proliferation. However, Bcl2 inhibition (by ABT737) reversed all the effects of SP600125. Our results suggested that DJ‐1 in PCa cells could promote the growth of PCa through autophagy inhibition, and JNK–Bcl2–Beclin1 signaling played an important role in it. The study provided new insights into the role of DJ‐1 in the development of PCa.     

Preconditioning Stimuli Induce Autophagy via Sphingosine Kinase 2 in Mouse Cortical Neurons

Sphingosine kinase 2 (SPK2) and autophagy are both involved in brain preconditioning, but whether preconditioning-induced SPK2 up-regulation and autophagy activation are linked mechanistically remains to be elucidated. In this study, we used in vitro and in vivo models to explore the role of SPK2-mediated autophagy in isoflurane and hypoxic preconditioning. In primary mouse cortical neurons, both isoflurane and hypoxic preconditioning induced autophagy. Isoflurane and hypoxic preconditioning protected against subsequent oxygen glucose deprivation or glutamate injury, whereas pretreatment with autophagy inhibitors (3-methyladenine or KU55933) abolished preconditioning-induced tolerance. Pretreatment with SPK2 inhibitors (ABC294640 and SKI-II) or SPK2 knockdown prevented preconditioning-induced autophagy. Isoflurane also induced autophagy in mouse in vivo as shown by Western blots for LC3 and p62, LC3 immunostaining, and electron microscopy. Isoflurane-induced autophagy in mice lacking the SPK1 isoform (SPK1^−/−), but not in SPK2^−/− mice. Sphingosine 1-phosphate and the sphingosine 1-phosphate receptor agonist FTY720 did not protect against oxygen glucose deprivation in cultured neurons and did not alter the expression of LC3 and p62, suggesting that SPK2-mediated autophagy and protections are not S1P-dependent. Beclin 1 knockdown abolished preconditioning-induced autophagy, and SPK2 inhibitors abolished isoflurane-induced disruption of the Beclin 1/Bcl-2 association. These results strongly indicate that autophagy is involved in isoflurane preconditioning both in vivo and in vitro and that SPK2 contributes to preconditioning-induced autophagy, possibly by disrupting the Beclin 1/Bcl-2 interaction.     

HIF-1 Regulation of Chondrocyte Apoptosis: Induction of the Autophagic Pathway

The goal of our investigation was to explore the mechanism by which hypoxia regulates growth plate chondrocyte survival. At low O2 tension, chondrocytes were refractory to a staurosporine (i.e., apoptosis-inducing) challenge. To determine whether hypoxic survival was due to the expression of HIF-1, we evaluated the response of HIF silenced cells to staurosporine. Both, silenced cells and control chondrocytes were equally sensitive to the apoptogen challenge. To learn if resistance was mediated by the proteins of the autophagic pathway, we examined the expression of Beclin 1 and LC3. Both proteins were present in the growth plate as well as in N1511 chondrocytes. Moreover, silencing of Beclin 1 resulted in enhanced chondrocyte death. Thus, this gene served to maintain chondrocyte survival activity. Besides serving a cytoprotective role, it is known that autophagy can function in cell death. Accordingly, to ascertain if autophagy might also sensitize cells to apoptosis, we activated autophagy and examined viability following exposure to an apoptogen. Treatment with the autophagy inhibitor 3-methyladenine rendered the chondrocytes refractory to killing, suggesting that sustained autophagy promoted cell death. We next examined expression of BID and caspase-8. When autophagy was suppressed, chondrocytes promoted caspase-8 activation and activated BID. Finally, we explored the relationship between HIF-1 and Beclin 1. We noted a decrease in Beclin 1 expression and loss of caspase-8 activation in HIF silenced cells and Beclin 1-Bcl-2 association was maintained upon serum starvation. This study indicates that HIF-1 serves to regulate both autophagy and apoptosis.     

Autophagy: an overlooked mechanism of HIV-1 pathogenesis and neuroAIDS?

Human immunodeficiency virus type 1 (HIV-1) establishes a persistent infection characterized by progressive depletion of CD4(+) lymphocytes and immunosuppression. Although extensive research has examined the importance of apoptosis as a cause of cell death associated with HIV-1 infection, the role of autophagy has been largely ignored. Our laboratory has examined the autophagic process in HIV-1-infected cells. Following infection of human peripheral blood CD4(+) T-cells or U937 cells with HIV-1 for 48 hours, the autophagy proteins Beclin 1 and LC3-II were found to be markedly decreased. Beclin 1 mRNA expression and autophagosomes were also reduced in HIV-1 infected cells. Thus, our data indicate that HIV-1 infection inhibits autophagy in infected cells in contrast to the previously described induction of autophagy by gp120 in uninfected bystander cells. It is likely that HIV-1 has evolved this mechanism as part of an elaborate attempt to evade the immune system while promoting its own replication. We believe that autophagy is an overlooked mechanism in HIV-1 pathogenesis and plays a particularly important role in the early cognitive impairment and dementia often associated with advanced AIDS. A model is presented that describes the potential role of autophagy in NeuroAIDS.     

Dendrimer-mediated siRNA delivery knocks down Beclin 1 and potentiates NMDA-mediated toxicity in rat cortical neurons

Autophagy is an important process which plays a key role in cellular homeostasis by degrading cytoplasmic components in the lysosomes, which facilitates recycling. Alterations to normal autophagy have been linked to excitotoxicity, but the mechanisms governing its signal transduction remain unclear. The aim of this study was to explore the role of autophagy in neuronal excitotoxic death by delivering small interfering RNA (siRNA) to rat cortical neurons, using a dendrimer to silence the autophagy-related gene 6 (beclin 1) and to determine the role of autophagy in excitotoxicity. We have found that the dendrimer is very efficient to deliver siRNA to rat cortical neurons, leading to almost complete removal of the target protein Beclin 1. In addition, NMDA increases autophagy markers, such as the protein levels of Beclin 1, the microtubule-associated light chain 3 (LC3) B-II/LC3B-I ratio, and monodansylcadaverine (MDC) labeling in rat cortical neurons. Moreover, NMDA also increases the formation of autophagosomes observed under a transmission electron microscope. Silencing beclin 1 expression blocked NMDA-induced autophagy. Moreover, Beclin 1 removal potentiated NMDA-induced neuronal death indicating that autophagy plays a protective role during excitotoxicity and suggesting that targeting autophagy might be a helpful therapeutic strategy in neurodegenerative diseases.     

HIF-1 regulation of chondrocyte apoptosis: induction of the autophagic pathway

The goal of our investigation was to explore the mechanism by which hypoxia regulates growth plate chondrocyte survival. At low O2 tension, chondrocytes were refractory to a staurosporine (i.e., apoptosis-inducing) challenge. To determine whether hypoxic survival was due to the expression of HIF-1, we evaluated the response of HIF silenced cells to staurosporine. Both, silenced cells and control chondrocytes were equally sensitive to the apoptogen challenge. To learn if resistance was mediated by the proteins of the autophagic pathway, we examined the expression of Beclin 1 and LC3. Both proteins were present in the growth plate as well as in N1511 chondrocytes. Moreover, silencing of Beclin 1 resulted in enhanced chondrocyte death. Thus, this gene served to maintain chondrocyte survival activity. Besides serving a cytoprotective role, it is known that autophagy can function in cell death. Accordingly, to ascertain if autophagy might also sensitize cells to apoptosis, we activated autophagy and examined viability following exposure to an apoptogen. Treatment with the autophagy inhibitor 3-methyladenine rendered the chondrocytes refractory to killing, suggesting that sustained autophagy promoted cell death. We next examined expression of BID and caspase-8. When autophagy was suppressed, chondrocytes promoted caspase-8 activation and activated BID. Finally, we explored the relationship between HIF-1 and Beclin 1. We noted a decrease in Beclin 1 expression and loss of caspase-8 activation in HIF silenced cells and Beclin 1-Bcl-2 association was maintained upon serum starvation. This study indicates that HIF-1 serves to regulate both autophagy and apoptosis.