BCL2/BCL-X(L) inhibition induces apoptosis, disrupts cellular calcium homeostasis, and prevents platelet activation.

Apoptosis in megakaryocytes results in the formation of platelets. The role of apoptotic pathways in platelet turnover and in the apoptotic-like changes seen after platelet activation is poorly understood. ABT-263 (Navitoclax), a specific inhibitor of antiapoptotic BCL2 proteins, which is currently being evaluated in clinical trials for the treatment of leukemia and other malignancies, induces a dose-limiting thrombocytopenia. In this study, the relationship between BCL2/BCL-X(L) inhibition, apoptosis, and platelet activation was investigated. Exposure to ABT-263 induced apoptosis but repressed platelet activation by physiologic agonists. Notably, ABT-263 induced an immediate calcium response in platelets and the depletion of intracellular calcium stores, indicating that on BCL2/BCL-X(L) inhibition platelet activation is abrogated because of a diminished calcium signaling. By comparing the effects of ABT-263 and its analog ABT-737 on platelets and leukemia cells from the same donor, we show, for the first time, that these BCL2/BCL-X(L) inhibitors do not offer any selective toxicity but induce apoptosis at similar concentrations in leukemia cells and platelets. However, reticulated platelets are less sensitive to apoptosis, supporting the hypothesis that treatment with ABT-263 induces a selective loss of older platelets and providing an explanation for the transient thrombocytopenia observed on ABT-263 treatment.

Desmethylclomipramine induces the accumulation of autophagy markers by blocking autophagic flux.

Alterations in the autophagic pathway are associated with the onset and progression of various diseases. However, despite the therapeutic potential for pharmacological modulators of autophagic flux, few such compounds have been characterised. Here we show that clomipramine, an FDA-approved drug long used for the treatment of psychiatric disorders, and its active metabolite desmethylclomipramine (DCMI) interfere with autophagic flux. Treating cells with DCMI caused a significant and specific increase in autophagosomal markers and a concomitant blockage of the degradation of autophagic cargo. This observation might be relevant in therapy in which malignant cells exploit autophagy to survive stress conditions, rendering them more susceptible to the action of cytotoxic agents. In accordance, DCMI-mediated obstruction of autophagic flux increased the cytotoxic effect of chemotherapeutic agents. Collectively, our studies describe a new function of DCMI that can be exploited for the treatment of pathological conditions in which manipulation of autophagic flux is thought to be beneficial.

Different pathways lead to mitochondrial fragmentation during apoptotic and excitotoxic cell death in primary neurons.

Mitochondrial fragmentation is recognized to be an important event during the onset of apoptosis. In this current study, we have used single cell imaging to investigate the role of the mitochondrial fission protein DRP-1 on mitochondrial morphology and mitochondrial fragmentation in primary hippocampal neurons undergoing necrotic or apoptotic cell death. Treatment of neurons with 500 nM staurosporine (apoptosis) or 30 µM glutamate (l-Glu; excitotoxic necrosis) produced a fragmentation and condensation of mitochondria, which although occurred over markedly different time frames appeared broadly similar in appearance. In neurons exposed to an apoptotic stimuli, inhibiting DRP-1 activity using overexpression of the dominant negative DRP-1(K38A) slowed the rate of mitochondrial fragmentation and decreased total cell death when compared to overexpression of wild-type DRP-1. In contrast, responses to l-Glu appeared DRP-1 independent. Similarly, alterations in the fission/fusion state of the mitochondrial network did not alter mitochondrial Ca(2+) uptake or the ability of l-Glu to stimulate excitotoxic Ca(2+) overload. Finally, apoptosis-induced mitochondrial fragmentation was observed concurrent with recruitment of Bax to the mitochondrial membrane. In contrast, during glutamate excitotoxicity, Bax remained in the cytosolic compartment. We conclude that different pathways lead to the appearance of fragmented mitochondria during necrotic and apoptotic neuronal cell death.

Mitochondrial Ca2+ signalling in hippocampal neurons.

High-resolution fluorescent imaging of mitochondrial-targeted probes was used to examine the ability of mitochondria to decode complex spatial and temporal Ca2+ signals evoked in synaptically active networks of hippocampal neurons. Green-to-red photoconversion of the mitochondrial-targeted probe, mito-Kaede, demonstrated that mitochondria were present as discrete organelles 2-6 microm in length. Real-time imaging of mitochondrial-targeted ratiometric pericam (2 mtRP) visualised rapid, repetitive, transient mitochondrial Ca2+ fluxes in response to periods of synaptic activation. Mitochondrial Ca2+ fluxes within cellular compartments were dependent on the extent of synaptic recruitment, but independent of cross-talk with the endoplasmic reticulum or the presence of an interconnected mitochondrial network. Mitochondria in dendritic regions demonstrated a greater sensitivity to synaptic activation compared with somatic mitochondria. Temporal decoding of synaptic signals was rate-limited by the activity of the mitochondrial Na+/Ca2+ exchanger. Spatial regulation of mitochondrial Ca2+ uptake was determined by the magnitude of the cytosolic Ca2+ rise in each cellular compartment.

Confocal microscopy: theory and applications for cellular signaling.

The development of confocal microscopy and the commercial availability of confocal microscopes have provided many laboratories with an extremely powerful approach to examine cellular structure and function. Allied with the development of suitable tools, it is now possible to interrogate a wide range of structural and functional aspects on both fixed and live cells. Here we describe the basic principles underlying confocal microscopy and provide methodological accounts of how it can be used to study aspects related particularly (but not exclusively) to the expression, activation, and regulation of signaling by G-protein-coupled receptors. Specifically we provide detailed protocols for examining: the cellular expression and distribution of proteins by immunocytochemistry; cytoplasmic and organelle Ca(2+) signaling using fluorescent indicators; second messenger generation using fluorescently tagged biosensors; and ligand/receptor internalization using fluorescently tagged peptide agonists and receptors.

Endoplasmic reticulum membrane reorganization is regulated by ionic homeostasis.

Recently we described a new, evolutionarily conserved cellular stress response characterized by a reversible reorganization of endoplasmic reticulum (ER) membranes that is distinct from canonical ER stress and the unfolded protein response (UPR). Apogossypol, a putative broad spectrum BCL-2 family antagonist, was the prototype compound used to induce this ER membrane reorganization. Following microarray analysis of cells treated with apogossypol, we used connectivity mapping to identify a wide range of structurally diverse chemicals from different pharmacological classes and established their ability to induce ER membrane reorganization. Such structural diversity suggests that the mechanisms initiating ER membrane reorganization are also diverse and a major objective of the present study was to identify potentially common features of these mechanisms. In order to explore this, we used hierarchical clustering of transcription profiles for a number of chemicals that induce membrane reorganization and discovered two distinct clusters. One cluster contained chemicals with known effects on Ca(2+) homeostasis. Support for this was provided by the findings that ER membrane reorganization was induced by agents that either deplete ER Ca(2+) (thapsigargin) or cause an alteration in cellular Ca(2+) handling (calmodulin antagonists). Furthermore, overexpression of the ER luminal Ca(2+) sensor, STIM1, also evoked ER membrane reorganization. Although perturbation of Ca(2+) homeostasis was clearly one mechanism by which some agents induced ER membrane reorganization, influx of extracellular Na(+) but not Ca(2+) was required for ER membrane reorganization induced by apogossypol and the related BCL-2 family antagonist, TW37, in both human and yeast cells. Not only is this novel, non-canonical ER stress response evolutionary conserved but so also are aspects of the mechanism of formation of ER membrane aggregates. Thus perturbation of ionic homeostasis is important in the regulation of ER membrane reorganization.

The in vitro cleavage of the hAtg proteins by cell death proteases.

It is becoming increasingly clear that there is crosstalk between the apoptotic and autophagic pathways, with autophagy helping to contribute to cell death by providing energy to allow the energy-requiring programmed cell death process to complete, as well as degrading cellular material in its own right. Recent evidence has suggested that Atg proteins can themselves be targets of caspases, providing potential regulation of autophagy as well as uncovering novel functions for fragments derived from Atg proteins. However, to date there has not been a detailed examination of which Atg proteins may be the targets of which death proteases. We show that the majority of human Atg (hAtg) proteins can be cleaved by calpain 1, which is activated in some apoptotic paradigms, as well as other forms of death. We also show that hAtg3 is cleaved by caspases-3, -6 and -8, hAtg6 (Beclin 1) is cleaved by caspase-3 and -6, while hAtg9, hAtg7 and the hAtg4 homologues can be cleaved by caspase-3. Cleavage of Beclin 1 was also seen in apoptosis of HeLa cells induced by staurosporine and TRAIL, along with cleavage of Atg3 and Atg4C. There were subtle effects of caspase inhibition on GFP-LC3 lipidation but more marked effects on the formation of GFP-LC3 puncta (a marker of autophagosome formation) and p62 degradation, indicating that caspase cleavage of autophagy-related proteins can affect the autophagic process. Notably we show that p62 is a target for caspase-6 and -8 cleavage.

Prevention of ischemic brain injury by treatment with the membrane penetrating apoptosis inhibitor, TAT-BH4.

In acute thromboembolic stroke, neurological damage is due to ischemia-induced apoptotic death of neuronal cells and the surrounding vascular network. Here, we demonstrate that the BH4 domain of the anti-apoptotic protein, Bcl-x(L), attached to the membrane transport peptide, TAT, reduces stroke injury after intracerebroventricular infusion into immature rats subjected to carotid artery ligation and additional exposure to hypoxia. The injected TAT-BH4 entered neuron bodies, maintained brain architecture, protected neuronal and endothelial cells from apoptosis and promoted neuronal stem cell recruitment. In vitro, TAT-BH4 enhanced the survival of endothelial cells exposed to H(2)O(2), increased neuronal differentiation, and induced axonal remodelling of adult neuronal stem cells. These findings indicate that TAT-BH4 administration protects against acute hypoxia/ischemia injury in the brain by preventing endothelial and neuron cell apoptosis and by inducing neuronal plasticity.

Hyperglycaemia inhibits Schwann cell proliferation and migration and restricts regeneration of axons and Schwann cells from adult murine DRG.

Poorly-controlled hyperglycaemia reduces peripheral nerve regeneration in diabetes through ill-understood mechanisms. Apoptosis is one proposed primary response. We examined how hyperglycaemia affects regeneration of axons and Schwann cells (SC) from cultured adult mouse Dorsal Root Ganglia (DRG) to separate cell-autonomous responses from systemic influences. Hyperglycaemia reduced neurite growth rate by 20-30% without altering growth cone density, indicating neuronal apoptosis was negligible. Moderate hyperglycaemia also profoundly retarded SC migration from DRG explants. This effect was independent of neuritogenesis and was reversible, indicating that SC had not died. In purified SC, even mild hyperglycaemia inhibited neuregulin-beta1-induced bromodeoxyuridine-incorporation and phosphorylation of retinoblastoma protein, indicating a block at the G1-S boundary. Moreover, migration of purified SC was inhibited by >90%. Thus, SC proliferation and migration, and axon regeneration from DRG neurons, are impaired by hyperglycaemia cell autonomously, while apoptosis is negligible. Impairment of these functions over time may exacerbate nerve injury-related diabetic neuropathy.

The dynamics of autophagy visualized in live cells: from autophagosome formation to fusion with endo/lysosomes.

Autophagy has been implicated in a range of disorders and hence is of major interest. However, imaging autophagy in real time has been hampered by lack of suitable markers. We have compared the potential of monodansylcadaverine, widely used as an autophagosomal marker, and the Atg8 homologue LC3, to follow autophagy by fluorescence microscopy whilst labelling late endosomes and lysosomes simultaneously using EGFP-CD63. Monodansylcadaverine labelled only acidic CD63-positive compartments in response to a range of autophagic inducers in various live or post-fixed cells, staining being identical in atg5(+/+) and atg5(-/-) MEFs in which autophagosome formation is disabled. Monodansylcadaverine staining was essentially indistinguishable from that of LysoTracker Red, LAMP-1 or LAMP-2. In contrast, 60-90% of EGFP-LC3-positive punctate organelles did not colocalise with LAMP-1/LAMP-2/CD63 and were monodansylcadaverine-negative while EGFP-LC3 puncta that did colocalise with LAMP-1/LAMP-2/CD63 were also monodansylcadaverine-positive. Hence monodansylcadaverine is no different from other markers of acidic compartments and it cannot be used to follow autophagosome formation. In contrast, fusion of mRFP-LC3-labelled autophagosomes with EGFP-CD63-positive endosomes and lysosomes and sequestration of dsRed-labelled mitochondria by EGFP-LC3- and EGFP-CD63-positive compartments could be visualized in real time. Moreover, transition of EGFP-LC3-I (45 kDa) to EGFP-LC3-II (43 kDa)-traced by immunoblotting and verified by [(3)H]ethanolamine labelling-revealed novel insights into the dynamics of autophagosome homeostasis, including the rapid activation of autophagy by the apoptotic inducer staurosporine prior to apoptosis proper. Use of fluorescent LC3 and a counter-fluorescent endosomal/lysosomal protein clearly allows the entire autophagic process to be followed by live cell imaging with high fidelity.

Osteonectin is a Schwann cell-secreted factor that promotes retinal ganglion cell survival and process outgrowth.

We have investigated the factors made by Schwann cells (SCs) that stimulate survival and neurite outgrowth from postnatal rat retinal ganglion cells (RGCs). These effects are preserved under K252a blockade of the Trk family of neurotrophin receptors and are not fully mimicked by the action of a number of known trophic factors. To identify novel factors responsible for this regenerative activity, we have used a radiolabelling assay. Proteins made by SCs were labelled radioactively and then fed to purified RGCs. The proteins taken up by the RGCs were then isolated and further characterized. Using this assay we have identified a major 40 kDa factor taken up by RGCs, which was microsequenced and shown to be the matricellular protein osteonectin (ON). Using an in vitro assay of purified RGCs we show that ON promotes both survival and neurite outgrowth. We conclude that ON has a potential new role in promoting CNS repair.

Factors secreted by Schwann cells stimulate the regeneration of neonatal retinal ganglion cells.

The adult mammalian central nervous system (CNS) does not repair after injury. However, we and others have shown in earlier work that the neonatal CNS is capable of repair and importantly of allowing regenerating axons to re-navigate through the same pathways as they did during development. This phase of neonatal repair is restricted by the fragility of neurons after injury and a lack of trophic factors that enable their survival. Our aim is to define better the factors that sustain neurons after injury and allow regeneration to occur. We describe some of our work using Schwann cells to promote the regeneration of neurons from young postnatal rodents. We have established rapid methods for purifying Schwann cells without the use of either anti-mitotic agents to suppress contaminating fibroblasts or mitotic stimulation to generate large numbers of Schwann cells. The rapidly purified Schwann cells have been used to generate conditioned medium that we have shown stimulates axon regeneration in cultured retinal ganglion cell neurons. We also show that the positive effects of Schwann cells are still present after pharmacological blockade of the neurotrophin receptors, suggesting that novel factors mediate these effects.

Sympathetic neuronal survival induced by retinal trophic factors.

Neuronal survival in the vertebrate peripheral nervous system depends on neurotrophic factors available from target tissues. In an attempt to identify novel survival factors, we have studied the effect of secreted factors from retinal cells on the survival of chick sympathetic ganglion neurons. Embryonic day 10 sympathetic neurons undergo programmed cell death after 48 h without appropriate levels of nerve growth factor (NGF). Retina Conditioned Media (RCM) from explants of embryonic day 11 retinas maintained for 4 days in vitro supported 90% of E10 chick sympathetic neurons after 48 h. Conditioned medium from purified chick retinal Muller glial cells supported nearly 100% of E10 chick sympathetic neurons. Anti-NGF (1 microg/mL) blocked the survival effect of NGF, but did not block the trophic effect of RCM. Neither BDNF nor NT4 (0.1-50 ng/mL) supported E10 sympathetic neuron survival. Incubation of chimeric immunoglobulin-receptors TrkA, TrkB, or TrkC had no effect on RCM-induced sympathetic neuron survival. The survival effects were not blocked by anti-GDNF, anti-TGFbeta, and anti-CNTF and were not mimicked by FGFb (0.1-10 nM). LY294002 at 50 microM, but not PD098059 blocked sympathetic survival induced by RCM. Further, the combination of RCM and NGF did not result in an increase in neuronal survival compared with NGF alone (82% survival after 48 h). The secreted factor in RCM is retained in subfractions with a molecular weight above 100 kDa, binds to heparin, and is unaffected by dialysis, but is heat sensitive. Our results indicate the presence of a high-molecular weight retinal secreted factor that supports sympathetic neurons in culture.