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.

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.

Guidelines for the use and interpretation of assays for monitoring autophagy.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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).