Obatoclax induces Atg7-dependent autophagy independent of beclin-1 and BAX/BAK

Direct pharmacological targeting of the anti-apoptotic B-cell lymphoma-2 (BCL-2) family is an attractive therapeutic strategy for treating cancer. Obatoclax is a pan-BCL-2 family inhibitor currently in clinical development. Here we show that, although obatoclax can induce mitochondrial apoptosis dependent on BCL-2 associated x protein/BCL-2 antagonist killer (BAX/BAK) consistent with its on-target pharmacodynamics, simultaneous silencing of both BAX and BAK did not abolish acute toxicity or loss of clonogenicity. This is despite complete inhibition of apoptosis. Obatoclax dramatically reduced viability without inducing loss of plasma membrane integrity. This was associated with rapid processing of light chain-3 (LC3) and reduction of S6 kinase phosphorylation, consistent with autophagy. Dramatic ultrastructural vacuolation, not typical of autophagy, was also induced. Silencing of beclin-1 failed to prevent LC3 processing, whereas knockout of autophagy-related (Atg)7 abolished LC3 processing but failed to prevent obatoclax-induced loss of clonogenicity or ultrastructural changes. siRNA silencing of Atg7 in BAX/BAK knockout mouse embryonic fibroblasts did not prevent obatoclax-induced loss of viability. Cells selected for obatoclax resistance evaded apoptosis independent of changes in BCL-2 family expression and displayed reduced LC3 processing. In summary, obatoclax exhibits BAX- and BAK-dependent and -independent mechanisms of toxicity and activation of autophagy. Mechanisms other than autophagy and apoptosis are blocked in obatoclax resistant cells and contribute significantly to obatoclax''s anticancer efficacy.     

Expression pattern and functions of autophagy-related gene atg5 in zebrafish organogenesis

The implications of autophagy-related genes in serious neural degenerative diseases have been well documented. However, the functions and regulation of the family genes in embryonic development remain to be rigorously studied. Here, we report on for the first time the important role of atg5 gene in zebrafish neurogenesis and organogenesis as evidenced by the spatiotemporal expression pattern and functional analysis. Using morpholino oligo knockdown and mRNA overexpression, we demonstrated that zebrafish atg5 is required for normal morphogenesis of brain regionalization and body plan as well as for expression regulation of neural gene markers: gli1, huC, nkx2.2, pink1, β-synuclein, xb51 and zic1. We further demonstrated that ATG5 protein is involved in autophagy by LC3-II/LC3I ratio and rapamycin-induction experiments, and that ATG5 is capable of regulating expression of itself gene in the manner of a feedback inhibition loop. In addition, we found that expression of another autophagy-related gene, atg12, is maintained at a higher constant level like a housekeeping gene. This indicates that the formation of the ATG12–ATG5 conjugate may be dependent on ATG5 protein generation and its splicing, rather than on ATG12 protein in zebrafish. Importantly, in the present study, we provide a mechanistic insight into the regulation and functional roles of atg5 in development of zebrafish nervous system.     

MCL1 is critical for mitochondrial function and autophagy in the heart

MCL1 (myeloid cell leukemia sequence 1 [BCL2-related]) is an anti-apoptotic BCL2 family protein that is upregulated in several human cancers. In malignancies, overexpression of MCL1 promotes cell survival and confers chemotherapeutic resistance. MCL1 is also highly expressed in normal myocardium, but the functional importance of MCL1 in myocytes has not been explored. We recently discovered that MCL1 plays an essential role in myocardial homeostasis and autophagy. Here, we discuss how loss of MCL1 in the adult mouse heart leads to mitochondrial dysfunction, impaired autophagy and development of heart failure.     

斑马鱼tbx2b调控心脏房室间隔发育的功能研究*

tbx2是早期心脏发育的关键基因。为进一步探究其对房室间隔(AVC)发育的影响,研究利用CRISPR/Cas9介导的基因敲除技术,成功构建了斑马鱼tbx2b突变体。通过T7E1酶切对其F₀进行敲除效率检测,结果显示平均敲除效率约为57.5%。F₁进一步筛选获得tbx2b杂合突变体,测序结果显示突变类型为11 bp碱基缺失的移码突变。tbx2b杂合子内交获得纯合子,tbx2b纯合突变体在5 dpf死亡并出现早期心脏环化异常表型。斑马鱼整胚原位杂交实验显示在3 dpf tbx2b纯合突变体中, 心脏腔室分化特异性标志基因nppa、nppb表达上调并异位表达在AVC,而AVC发育关键基因has2的表达消失。高效构建tbx2b突变体并初探其对下游基因的影响,为后续深入研究tbx2b对心脏AVC发育的作用奠定了基础,同时加深了人们对早期心脏调控网络的认识。     

Cascade effect of cardiac myogenesis gene expression during cardiac looping in <Emphasis Type="Italic">tbx5</Emphasis> knockdown zebrafish embryos

Zebrafish tbx5 expresses in the heart, pectoral fins and eyes of zebrafish during embryonic development. In zebrafish, injection of tbx5 morpholino antisense RNA caused changes of heart conformation, defect of heart looping, pericardium effusion, dropsy of ventral position and decreased heart rate. We suggested that cardiac myogenesis genes might be responsible for this phenomenon. Morpholino antisense RNA which against the initiation site of tbx5 gene was designed in order to knockdown the expression of tbx5, and the results were analyzed by whole-mount in situ hybridization and quantitative real-time PCR. Expression of cardiac myogenesis genes amhc, vmhc and cmlc2 were expressed constantly at the early embryonic development and reached its highest rate right before cardiac looping initiated. These cardiac myogenesis genes showed insufficient expressions within different heart defect embryos. Moreover, vmhc showed ectopic expression in addition to heart looping defect in heart defective embryos at 36 hpf. Our data suggests that the heart failure caused by the knockdown of tbx5 gene might result from the down-regulation of cardiac myogenesis genes. Jen Her Lu and Jenn Kan Lu contributed equally to this work.     

Coupling in vitro and in vivo paradigm reveals a dose dependent inhibition of angiogenesis followed by initiation of autophagy by C6-ceramide

The activity of N-hexanoyl-D-erythro-sphingosine, a C6-ceramide against angiogenesis was tested in vitro and in vivo. The effect of ceramide in inhibiting MCF-7 cancer cells was also determined. The aim of this study was to potentiate the effect of ceramide as anti-angiogenic compound that can regulate tumor induced angiogenesis.C6-ceramide inhibited vascular endothelial growth factor (VEGF)-induced human umbilical vein endothelial cells (HUVEC) tube formation in a dose-dependent manner within 24 hours. Ceramide at concentrations between 12.5 and 25 μM inhibited the viability of MCF-7 cells and reduced VEGF-induced cell migration in 24 hours. At 50 μM, ceramide induced MCF-7 cell death via autophagy as demonstrated by accumulation of MDC in ceramide-treated MCF-7 vacuoles. The expression of VEGF was reduced and the levels of cathepsin D in MCF-7 increased. In vivo, 50 μM ceramide caused a 40% reduction of new vessel formation in the CAM assay within 24 hours. Zebrafish exposed to 100 - 400 μM ceramide had a distinct disruption of blood vessel development at 48 hours post-fertilization. Ceramide-exposed embryos also had primary motoneurons exhibiting abnormal axonal trajectories and ectopic branching. Ceramide induced cell-death was not detected in the zebrafish assay. Collectively, these data indicate that ceramide is a potent anti-angiogenic compound and that the mechanism underlying its anti-angiogenic capabilities does not rely upon the induction of apoptosis.     

Autophagy deficiency leads to accumulation of ubiquitinated proteins,ER stress,and cell death in Arabidopsis

Autophagy is a homeostatic degradation and recycling process that is also involved in defense against microbial pathogens and in certain forms of cellular suicide. Autophagy has been proposed to negatively regulate plant immunity-associated cell death related to the hypersensitive response (HR), as older autophagy-deficient mutants are unable to contain this type of cell death 5 to 10 d after infection. Such spreading cell death was found to require NPR1 (nonexpressor of PR genes 1), but surprisingly did not occur in younger atg mutants. In contrast, we find that npr1 mutants are not impaired in rapid programmed cell death activation upon pathogen recognition. Furthermore, our molecular evidence suggests that the NPR1-dependent spreading cell death in older atg mutants may originate from an inability to cope with excessive accumulation of ubiquitinated proteins and ER stress which derive from salicylic acid (SA)-dependent signaling (e.g., systemic acquired resistance). We also demonstrate that both senescence and immunity-related cell death seen in older atg mutants can be recapitulated in younger atg mutants primed with ER stress. We therefore propose that the reduction in SA signaling caused by npr1 loss-of-function is sufficient to alleviate the stress levels accumulated during aging in autophagy deficient cells which would otherwise become insurmountable and lead to uncontrolled cell death.     

Induction of apoptosis and inhibition of cell growth by <Emphasis Type="Italic">tbx5</Emphasis> knockdown contribute to dysmorphogenesis in Zebrafish embryos

Background  

The tbx5 mutation in human causes Holt-Oram syndrome, an autosomal dominant condition characterized by a familial history of congenital heart defects and preaxial radial upper-limb defects. We report aberrant apoptosis and dormant cell growth over head, heart, trunk, fin, and tail of zebrafish embryos with tbx5 deficiency correspond to the dysmorphogenesis of tbx5 morphants.     

Autophagy is required for ectoplasmic specialization assembly in sertoli cells

The ectoplasmic specialization (ES) is essential for Sertoli-germ cell communication to support all phases of germ cell development and maturity. Its formation and remodeling requires rapid reorganization of the cytoskeleton. However, the molecular mechanism underlying the regulation of ES assembly is still largely unknown. Here, we show that Sertoli cell-specific disruption of autophagy influenced male mouse fertility due to the resulting disorganized seminiferous tubules and spermatozoa with malformed heads. In autophagy-deficient mouse testes, cytoskeleton structures were disordered and ES assembly was disrupted. The disorganization of the cytoskeleton structures might be caused by the accumulation of a negative cytoskeleton organization regulator, PDLIM1, and these defects could be partially rescued by Pdlim1 knockdown in autophagy-deficient Sertoli cells. Altogether, our works reveal that the degradation of PDLIM1 by autophagy in Sertoli cells is important for the proper assembly of the ES, and these findings define a novel role for autophagy in Sertoli cell-germ cell communication.