Luc Leybaert

The gap junction cellular internet: connexin hemichannels enter the signalling limelight

Cxs (connexins), the protein subunits forming gap junction intercellular communication channels, are transported to the plasma membrane after oligomerizing into hexameric assemblies called connexin hemichannels (CxHcs) or connexons, which dock head-to-head with partner hexameric channels positioned on neighbouring cells. The double membrane channel or gap junction generated directly couples the cytoplasms of interacting cells and underpins the integration and co-ordination of cellular metabolism, signalling and functions, such as secretion or contraction in cell assemblies. In contrast, CxHcs prior to forming gap junctions provide a pathway for the release from cells of ATP, glutamate, NAD+ and prostaglandin E2, which act as paracrine messengers. ATP activates purinergic receptors on neighbouring cells and forms the basis of intercellular Ca2+ signal propagation, complementing that occuring more directly via gap junctions. CxHcs open in response to various types of external changes, including mechanical, shear, ionic and ischaemic stress. In addition, CxHcs are influenced by intracellular signals, such as membrane potential, phosphorylation and redox status, which translate external stresses to CxHc responses. Also, recent studies demonstrate that cytoplasmic Ca2+ changes in the physiological range act to trigger CxHc opening, indicating their involvement under normal non-pathological conditions. CxHcs not only respond to cytoplasmic Ca2+, but also determine cytoplasmic Ca2+, as they are large conductance channels, suggesting a prominent role in cellular Ca2+ homoeostasis and signalling. The functions of gap-junction channels and CxHcs have been difficult to separate, but synthetic peptides that mimic short sequences in the Cx subunit are emerging as promising tools to determine the role of CxHcs in physiology and pathology.

Engaging neuroscience to advance translational research in brain barrier biology

The delivery of many potentially therapeutic and diagnostic compounds to specific areas of the brain is restricted by brain barriers, of which the most well known are the blood–brain barrier (BBB) and the blood–cerebrospinal fluid (CSF) barrier. Recent studies have shown numerous additional roles of these barriers, including an involvement in neurodevelopment, in the control of cerebral blood flow, and — when barrier integrity is impaired — in the pathology of many common CNS disorders such as Alzheimers disease, Parkinsons disease and stroke.

Intracellular calcium changes trigger connexin 32 hemichannel opening

Connexin hemichannels have been proposed as a diffusion pathway for the release of extracellular messengers like ATP and others, based on connexin expression models and inhibition by gap junction blockers. Hemichannels are opened by various experimental stimuli, but the physiological intracellular triggers are currently not known. We investigated the hypothesis that an increase of cytoplasmic calcium concentration ([Ca2+]i) triggers hemichannel opening, making use of peptides that are identical to a short amino‐acid sequence on the connexin subunit to specifically block hemichannels, but not gap junction channels. Our work performed on connexin 32 (Cx32)‐expressing cells showed that an increase in [Ca2+]i triggers ATP release and dye uptake that is dependent on Cx32 expression, blocked by Cx32 (but not Cx43) mimetic peptides and a calmodulin antagonist, and critically dependent on [Ca2+]i elevation within a window situated around 500 nM. Our results indicate that [Ca2+]i elevation triggers hemichannel opening, and suggest that these channels are under physiological control.

Connexin-related signaling in cell death: to live or let die?

Evidence is accumulating that some forms of cell death, like apoptosis, are not only governed by the complex interplay between extracellular and intracellular signals but are also strongly influenced by intercellular communicative networks. The latter is provided by arrays of channels consisting of connexin proteins, with gap junctions directly connecting the cytoplasm of neighboring cells and hemichannels positioned as pores that link the cytoplasm to the extracellular environment. The role of gap junctions in cell death communication has received considerable interest and recently hemichannels have joined in as potentially toxic pores adding their part to the cell death process. However, despite a large body of existing evidence, especially for gap junctions, the exact contribution of the connexin channel family still remains controversial, as both gap junctions and hemichannels may furnish cell death as well as cell survival signals. An additional layer of complexity is formed by the fact that connexin proteins as such, beyond their channel function, may influence the cell death process. We here review the current knowledge on connexins and their channels in cell death and specifically address the molecular mechanisms that underlie connexin-related signaling. We also briefly focus on pannexins, a novel set of connexin-like proteins that have been implicated in cellular responses to pathological insults.

Reduced amounts and abnormal forms of phospholipase C zeta (PLCzeta) in spermatozoa from infertile men.

BACKGROUND In mammals, oocyte activation at fertilization is thought to be induced by the sperm-specific phospholipase C zeta (PLCzeta). However, it still remains to be conclusively shown that PLCzeta is the endogenous agent of oocyte activation. Some types of human infertility appear to be caused by failure of the sperm to activate and this may be due to specific defects in PLCzeta. METHODS AND RESULTS Immunofluorescence studies showed PLCzeta to be localized in the equatorial region of sperm from fertile men, but sperm deficient in oocyte activation exhibited no specific signal in this same region. Immunoblot analysis revealed reduced amounts of PLCzeta in sperm from infertile men, and in some cases, the presence of an abnormally low molecular weight form of PLCzeta. In one non-globozoospermic case, DNA analysis identified a point mutation in the PLCzeta gene that leads to a significant amino acid change in the catalytic region of the protein. Structural modelling suggested that this defect may have important effects upon the structure and function of the PLCzeta protein. cRNA corresponding to mutant PLCzeta failed to induce calcium oscillations when microinjected into mouse oocytes. Injection of infertile human sperm into mouse oocytes failed to activate the oocyte or trigger calcium oscillations. Injection of such infertile sperm followed by two calcium pulses, induced by assisted oocyte activation, activated the oocytes without inducing the typical pattern of calcium oscillations. CONCLUSIONS Our findings illustrate the importance of PLCzeta during fertilization and suggest that mutant forms of PLCzeta may underlie certain types of human male infertility.

Gap junctions and the propagation of cell survival and cell death signals

Gap junctions are a unique type of intercellular channels that connect the cytoplasm of adjoining cells. Each gap junction channel is comprised of two hemichannels or connexons and each connexon is formed by the aggregation of six protein subunits known as connexins. Gap junction channels allow the intercellular passage of small (< 1.5 kDa) molecules and regulate essential processes during development and differentiation. However, their role in cell survival and cell death is poorly understood. We review experimental data that support the hypothesis that gap junction channels may propagate cell death and survival modulating signals. In addition, we explore the hypothesis that hemichannels (or unapposed connexons) might be used as a paracrine conduit to spread factors that modulate the fate of the surrounding cells. Finally, direct signal transduction activity of connexins in cell death and survival pathways is addressed.

Ca2+ regulation of connexin 43 hemichannels in C6 glioma and glial cells

Connexin hemichannels have a low open probability under normal conditions but open in response to various stimuli, forming a release pathway for small paracrine messengers. We investigated hemichannel-mediated ATP responses triggered by changes of intracellular Ca(2+) ([Ca(2+)](i)) in Cx43 expressing glioma cells and primary glial cells. The involvement of hemichannels was confirmed with gja1 gene-silencing and exclusion of other release mechanisms. Hemichannel responses were triggered when [Ca(2+)](i) was in the 500nM range but the responses disappeared with larger [Ca(2+)](i) transients. Ca(2+)-triggered responses induced by A23187 and glutamate activated a signaling cascade that involved calmodulin (CaM), CaM-dependent kinase II, p38 mitogen activated kinase, phospholipase A2, arachidonic acid (AA), lipoxygenases, cyclo-oxygenases, reactive oxygen species, nitric oxide and depolarization. Hemichannel responses were also triggered by activation of CaM with a Ca(2+)-like peptide or exogenous application of AA, and the cascade was furthermore operational in primary glial cells isolated from rat cortex. In addition, several positive feed-back loops contributed to amplify the responses. We conclude that an elevation of [Ca(2+)](i) triggers hemichannel opening, not by a direct action of Ca(2+) on hemichannels but via multiple intermediate signaling steps that are adjoined by distinct signaling mechanisms activated by high [Ca(2+)](i) and acting to restrain cellular ATP loss.

Neurobarrier Coupling in the Brain: A Partner of Neurovascular and Neurometabolic Coupling?:

Neurovascular and neurometabolic coupling help the brain to maintain an appropriate energy flow to the neural tissue under conditions of increased neuronal activity. Both coupling phenomena provide us, in addition, with two macroscopically measurable parameters, blood flow and intermediate metabolite fluxes, that are used to dynamically image the functioning brain. The main energy substrate for the brain is glucose, which is metabolized by glycolysis and oxidative breakdown in both astrocytes and neurons. Neuronal activation triggers increased glucose consumption and glucose demand, with new glucose being brought in by stimulated blood flow and glucose transport over the blood-brain barrier. Glucose is shuttled over the barrier by the GLUT-1 transporter, which, like all transporter proteins, has a ceiling above which no further stimulation of the transport is possible. Blood-brain barrier glucose transport is generally accepted as a nonrate-limiting step but to prevent it from becoming rate-limiting under conditions of neuronal activation, it might be necessary for the transport parameters to be adapted to the increased glucose demand. It is proposed that the blood-brain barrier glucose transport parameters are dynamically adapted to the increased glucose needs of the neural tissue after activation according to a neurobarrier coupling scheme. This review presents neurobarrier coupling within the current knowledge on neurovascular and neurometabolic coupling, and considers arguments and evidence in support of this hypothesis.

Inositol-trisphosphate-dependent intercellular calcium signaling in and between astrocytes and endothelial cells.

Interactions between astrocytes and endothelial cells are believed to play an important role in the control of blood‐brain barrier permeability and transport. Astrocytes and endothelial cells respond to a variety of stimuli with an increase of intracellular free calcium ([Ca2+]i) that is propagated to adjacent cells as an intercellular Ca2+ wave. We hypothesized that intercellular Ca2+ signaling also occurs between astrocytes and endothelial cells, and we investigated this possibility in co‐cultures of primary astrocytes and an endothelial cell line using caged messengers. Intercellular Ca2+ waves, induced by mechanical stimulation of a single cell, propagated from astrocytes to endothelial cells and vice versa. Intercellular Ca2+ waves could also be induced by flash photolysis of pressure‐injected caged inositol trisphosphate (IP3) and also by applying the flash to remote noninjected cells. Ca2+ waves induced by flash photolysis propagated from endothelial cells to astrocytes but not from astrocytes to endothelial cells even though caged IP3 diffused between the two cell types. Flash photolysis of caged Ca2+ (NP‐EGTA) resulted in an increase of [Ca2+]i but did not initiate an intercellular Ca2+ wave. We conclude that an increase of IP3 in a single cell is sufficient to initiate an intercellular Ca2+ wave that is propagated by the diffusion of IP3 to neighboring cells and that can be communicated between astrocytes and endothelial cells in co‐culture. By contrast, Ca2+ diffusion via gap junctions does not appear to be sufficient to propagate an intercellular Ca2+ wave. We suggest that intercellular Ca2+ waves may play a role in astrocyte‐endothelial interactions at the blood‐brain barrier. GLIA 24:398–407, 1998.

Intercellular Ca2+ Waves: Mechanisms and Function

Intercellular calcium (Ca(2+)) waves (ICWs) represent the propagation of increases in intracellular Ca(2+) through a syncytium of cells and appear to be a fundamental mechanism for coordinating multicellular responses. ICWs occur in a wide diversity of cells and have been extensively studied in vitro. More recent studies focus on ICWs in vivo. ICWs are triggered by a variety of stimuli and involve the release of Ca(2+) from internal stores. The propagation of ICWs predominately involves cell communication with internal messengers moving via gap junctions or extracellular messengers mediating paracrine signaling. ICWs appear to be important in both normal physiology as well as pathophysiological processes in a variety of organs and tissues including brain, liver, retina, cochlea, and vascular tissue. We review here the mechanisms of initiation and propagation of ICWs, the key intra- and extracellular messengers (inositol 1,4,5-trisphosphate and ATP) mediating ICWs, and the proposed physiological functions of ICWs.