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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 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.

Canonical endoplasmic reticulum (ER) stress, which occurs in many physiological and disease processes, results in activation of the unfolded protein response (UPR). We now describe a new, evolutionarily conserved cellular stress response characterised by a striking, but reversible, reorganisation of ER membranes that occurs independently of the UPR, resulting in impaired ER transport and function. This reorganisation is characterised by a dramatic redistribution and clustering of ER membrane proteins. ER membrane aggregation is regulated, in part, by anti-apoptotic BCL-2 family members, particularly MCL-1. Using connectivity mapping, we report the widespread occurrence of this stress response by identifying several structurally diverse chemicals from different pharmacological classes, including antihistamines, antimalarials and antipsychotics, which induce ER membrane reorganisation. Furthermore, we demonstrate the potential of ER membrane aggregation to result in pathological consequences, such as the long-QT syndrome, a cardiac arrhythmic abnormality, arising because of a novel trafficking defect of the human ether-a-go-go-related channel protein from the ER to the plasma membrane. Thus, ER membrane reorganisation is a feature of a new cellular stress pathway, clearly distinct from the UPR, with important consequences affecting the normal functioning of the ER.

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

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.

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.

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., (Copyright 2010 Wiley Periodicals, Inc.)

Our previous study identified osteonectin (ON) in a screen of factors made by Schwann cells (SCs) which promoted peripheral and central neurons survival and neuritogenesis, however, the mechanisms of ON promoting effects are largely unknown. In the present study, we investigated the effects of ON-deficient SC-conditioned medium (SCCM) and molecular mechanisms of ON, in regulating retinal ganglion cells (RGCs) survival and neurite outgrowth. Neonatal rat RGCs and SCs were purified by immunopanning technique. RGC survival and neuritogenesis reduced significantly when treated with either ON-null mice SCCM or ON-immunodepleted (IP) SCCM (P<0.05). In contrast to wild type SCCM, in the presence of a tyrosine kinase receptor (Trk) inhibitor (K252a), ON-null mice SCCM-induced neuritogenesis were further reduced by 24%. The Trk-mediated signaling pathways became more sensitive to K252a inhibition in the absence of ON. We also showed the synergistic effects of ON and brain-derived neurotrophic factor (BDNF) in promoting RGCs growth and the involvement of ON in two major neurotrophin-mediated signaling pathways, PI-3K-Akt and MAPK-Erk1/2. ON alone activated Akt phosphorylation and increased survival. Blockage of TrkB signalling pathway by TrkB-Fc chimera (BDNF scavenger) or K252a in ON-treated cultures reduced Akt-P level significantly. This suggests that ON induces BDNF synthesis and secretion from RGCs. The enhancement of neuritogenesis and Erk1/2 phosphorylation by ON in BDNF-treated cultures further demonstrate the signaling pathways responsible for the synergistic effect of ON on BDNF-induced neurite outgrowth. To the best of our knowledge, this is the first report showing the synergistic effects of ON on classical neurotrophins which participate in the same signalling pathways in regulating RGC neurite outgrowth.

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.

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.

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.

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.

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.

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.

We have used PC12 cells to examine the effects of factors secreted by Schwann cells that promote cell survival and neurite outgrowth, and hence are likely candidates for promoting neuronal regeneration. RT-PCR showed that primary Schwann cells produced a range of neurotrophins, excluding NT3, but this profile was different from either of two cell lines SCTM41 or PVGSCSV40T, or forskolin-expanded Schwann cells. The effects of Schwann cell conditioned media on neurite outgrowth was tested against a range of factors, and showed clear neuritogenic effects. Of the factors tested, only NGF had a significant response on neuritogenesis. Western blotting for neurofilaments showed that primary Schwann cells induced a strong response close to that of NGF. The Trk tyrosine kinase inhibitor K252a did not block the neuritogenic effects of primary Schwann cells. In contrast, K252a blocked both NGF and the SCTM41 cell effects. Schwann cell conditioned media also enhanced PC12 cell survival. Again, in contrast with NGF or SCTM41 cells, the primary Schwann cell effect was Trk tyrosine kinase independent. The Schwann cell conditioned medium contains a protein factor (greater than 12 kDa and broken down by trypsin treatment) with remarkable thermal stability (unaffected at 95 degrees C for 15 min) and the ability to bind heparin. Our results provide clear evidence that Schwann cells produce factors other than those already known to stimulate a neural phenotype in PC12 cells, and which thus have potential regeneration enhancing effects., (Copyright (c) 2005 Wiley Periodicals, Inc.)

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.

Transgenic or knockout mouse models provide the opportunity to study the function of disease-related or novel genes. However, a confounding factor in all such research is the genetic and phenotypic variation of the mouse strain used to construct the models. A trait which is frequently studied in transgenic models of neurological disorders is synaptic transmission and plasticity of the dentate gyrus of the hippocampus. Consequently, we have investigated the variation in this trait across five strains of mouse (129 Ola, C3H, C57 albino, DBA/2, and FVB/N), in vivo. 129 Ola mice were found to have significantly larger maximal evoked EPSP slope and population spike amplitudes compared to the other strains. No differences across strains were found in paired-pulse facilitation of EPSP slope, a measure of pre-synaptic short-term plasticity. DBA/2 mice showed significantly reduced paired-pulse inhibition of population spike, a measure of poly-synaptic inhibitory feedback within the dentate gyrus. Potentiation of EPSP and population spike, following tetanic stimulation of the perforant path, was observed in all strains. However, DBA/2 mice showed a deficit in the maintenance of potentiation over 1 h, which confirms a previous report [S. Matsuyama, U. Namgung, A. Routtenberg, Long-term potentiation persistence greater in C57BL/6 than DBA/2 mice: predicted on basis of protein kinase C levels and learning performance, Brain Res. 763 (1997) 127-130]. These results show that electrophysiological traits do vary significantly across mouse strains, and that the selection of the strain may have a significant impact on results. Furthermore, since production of a transgenic or knock-out mouse frequently requires cross-breeding, care should be taken in establishing the contribution of parent strains to the final phenotype, as well as the potential interaction with the phenotype arising from the knock-out or transgene.

The subtelomeric regions of the chromosomes of many organisms contain gene families that allow adaptation to a changing environment. In a number of parasites, these subtelomeric gene families encode cell surface proteins that undergo antigenic variation. Proteases are another important virulence determinant in pathogenic microorganisms. We report the localization of the PRT1 protease of the pathogenic fungus Pneumocystis carinii sp. f. carinii, encoded by a subtelomeric gene family, to the cell surface of both the trophozoite and the cyst phase of the organism. Using anti-PRT1 antiserum, we demonstrated specificity to P. carinii sp. f. carinii in sections of infected rat lungs and, using immunofluorescence, we showed that the PRT1 protease has the characteristic distribution of a surface protein. The anti-PRT1 antiserum showed cross-reactivity with a number of P. carinii sp. f. carinii proteins migrating between 185 kDa and 28 kDa, the majority migrating between 42 kDa and 52 kDa, a region that has been shown by serological studies to contain important immunodominant P. carinii proteins. Cross-reactivity was also observed with P. carinii sp. f. hominis proteins. We have also cloned a portion of the catalytic domain of PRT1 from P. carinii sp. f. hominis, P. carinii sp. f. muris and P. carinii sp. f. rattus. Our data suggest that the PRT1 protease plays an important role in the pathogenicity of P. carinii.