Randy F. Stout

Glial cells in (patho)physiology.

J. Neurochem. (2012) 121, 4–27.

Connexins modulate autophagosome biogenesis

The plasma membrane contributes to the formation of autophagosomes, the double-membrane vesicles that sequester cytosolic cargo and deliver it to lysosomes for degradation during autophagy. In this study, we have identified a regulatory role for connexins (Cx), the main components of plasma membrane gap junctions, in autophagosome formation. We have found that plasma-membrane-localized Cx proteins constitutively downregulate autophagy through a direct interaction with several autophagy-related proteins involved in the initial steps of autophagosome formation, such as Atg16 and components of the PI(3)K autophagy initiation complex (Vps34, Beclin-1 and Vps15). On nutrient starvation, this inhibitory effect is released by the arrival of Atg14 to the Cx–Atg complex. This promotes the internalization of Cx–Atg along with Atg9, which is also recruited to the plasma membrane in response to starvation. Maturation of the Cx-containing pre-autophagosomes into autophagosomes leads to degradation of these endogenous inhibitors, allowing for sustained activation of autophagy.

Single-vesicle architecture of synaptobrevin2 in astrocytes

Exocytic transmitter release is regulated by the SNARE complex, which contains a vesicular protein, synaptobrevin2 (Sb2). However, Sb2 vesicular arrangement is unclear. Here we use super-resolution fluorescence microscopy to study the prevalence and distribution of endogenous and exogenous Sb2 in single vesicles of astrocytes, the most abundant glial cells in the brain. We tag Sb2 protein at C- and N termini with a pair of fluorophores, which allows us to determine the Sb2 length and geometry. To estimate total number of Sb2 proteins per vesicle and the quantity necessary for the formation of fusion pores, we treat cells with ATP to stimulate Ca2+-dependent exocytosis, increase intracellular alkalinity to enhance the fluorescence presentation of yellow-shifted pHluorin (YpH), appended to the vesicle lumen domain of Sb2, and perform photobleaching of YpH fluorophores. Fluorescence intensity analysis reveals that the total number of endogenous Sb2 units or molecules per vesicle is ≤25.

Nanopore sensing of botulinum toxin type B by discriminating an enzymatically cleaved Peptide from a synaptic protein synaptobrevin 2 derivative.

Botulinum neurotoxins (BoNTs) are the most lethal toxin known to human. Biodefense requires early and rapid detection of BoNTs. Traditionally, BoNTs can be detected by looking for signs of botulism in mice that receive an injection of human material, serum or stool. While the living animal assay remains the most sensitive approach, it is costly, slow and associated with legal and ethical constrains. Various biochemical, optical and mechanical methods have been developed for BoNTs detection with improved speed, but with lesser sensitivity. Here, we report a novel nanopore-based BoNT type B (BoNT-B) sensor that monitors the toxin’s enzymatic activity on its substrate, a recombinant synaptic protein synaptobrevin 2 derivative. By analyzing the modulation of the pore current caused by the specific BoNT-B-digested peptide as a marker, the presence of BoNT-B at a subnanomolar concentration was identified within minutes. The nanopore detector would fill the niche for a much needed rapid and highly sensitive detection of neurotoxins, and provide an excellent system to explore biophysical mechanisms for biopolymer transportation.

Comparative analysis of optogenetic actuators in cultured astrocytes.

Highlights • We compared six optogenetic tools to selectively stimulate and study astrocytes.• Channelrhodopsin-2 variants cause release of Ca2+ from intracellular stores.• Opto-GPCRs activate selective second messenger cascades, leading to [Ca2+]i rises.• Autocrine action of ATP mediates the bulk of [Ca2+]i signals evoked by opto-GPCRs.• Current optogenetic tools initiate relevant signalling events in astrocytes.

Caenorhabditis elegans glia modulate neuronal activity and behavior.

Glial cells of Caenorhabditis elegans can modulate neuronal activity and behavior, which is the focus of this review. Initially, we provide an overview of neuroglial evolution, making a comparison between C. elegans glia and their genealogical counterparts. What follows is a brief discussion on C. elegans glia characteristics in terms of their exact numbers, germ layers origin, their necessity for proper development of sensory organs, and lack of their need for neuronal survival. The more specific roles that various glial cells have on neuron-based activity/behavior are succinctly presented. The cephalic sheath glia are important for development, maintenance and activity of central synapses, whereas the amphid glia seem to set the tone of sensory synapses; these glial cell types are ectoderm-derived. Mesoderm-derived Glial-Like cells in the nerve Ring (GLRs) appear to be a part of the circuit for production of motor movement of the worm anterior. Finally, we discuss tools and approaches utilized in studying C. elegans glia, which are assets available for this animal, making it an appealing model, not only in neurosciences, but in biology in general.

Connexin type and fluorescent protein fusion tag determine structural stability of gap junction plaques

Background: Connexin proteins form gap junction channel clusters termed plaques. Results: Microscopy with fluorescent protein-tagged connexins showed that connexin26 and connexin30 plaque arrangement is dynamic, but gap junctions of connexin43 are stabilized by its C-terminal region. Conclusion: The arrangement of channels in gap junctions depends on connexin type. Significance: These findings help to clarify how gap junction plaque dynamics and composition could modulate intercellular communication. Gap junctions (GJs) are made up of plaques of laterally clustered intercellular channels and the membranes in which the channels are embedded. Arrangement of channels within a plaque determines subcellular distribution of connexin binding partners and sites of intercellular signaling. Here, we report the discovery that some connexin types form plaque structures with strikingly different degrees of fluidity in the arrangement of the GJ channel subcomponents of the GJ plaque. We uncovered this property of GJs by applying fluorescence recovery after photobleaching to GJs formed from connexins fused with fluorescent protein tags. We found that connexin 26 (Cx26) and Cx30 GJs readily diffuse within the plaque structures, whereas Cx43 GJs remain persistently immobile for more than 2 min after bleaching. The cytoplasmic C terminus of Cx43 was required for stability of Cx43 plaque arrangement. We provide evidence that these qualitative differences in GJ arrangement stability reflect endogenous characteristics, with the caveat that the sizes of the GJs examined were necessarily large for these measurements. We also uncovered an unrecognized effect of non-monomerized fluorescent protein on the dynamically arranged GJs and the organization of plaques composed of multiple connexin types. Together, these findings redefine our understanding of the GJ plaque structure and should be considered in future studies using fluorescent protein tags to probe dynamics of highly ordered protein complexes.

A Caenorhabditis elegans locomotion phenotype caused by transgenic repeats of the hlh-17 promoter sequence.

Transgene technology is one of the most heavily relied upon tools in modern biological research. Expression of an exogenous gene within cells, for research and therapeutic applications, nearly always includes promoters and other regulatory sequences. We found that repeats of a non-protein coding transgenic sequence produced profound changes to the behavior of the nematode Caenorhabditis elegans. These changes were produced by a glial promoter sequence but, unexpectedly, major deficits were observed specifically in backward locomotion, a neuron-driven behavior. We also present evidence that this behavioral phenotype is transpromoter copy number-dependent and manifests early in development and is maintained into adulthood of the worm.

Adrenergic Receptors on Astrocytes Modulate Gap Junctions

Abstract Adrenergic receptors in astrocytes have both direct and indirect impact on intercellular communication in the nervous system. Direct mechanisms include activation of second messenger pathways that produce posttranslational modifications of the gap junction proteins [connexins (Cxs)] or activate transcription factors or other processes such as miRNAs that dictate or modify Cx gene expression or protein translation. These direct effects change the strength of intercellular communication through modifying open probability of the gap junction channels, altering balance between delivery to the membrane and degradation or through regulation of transcript abundance. Indirect actions with potentially profound implications for glial and neuronal network activity include alterations in the intracellular concentration of metabolites, energy-supplying molecules and second messengers, thereby changing the diffusional driving force responsible for intercellular spread. Although direct effects target the gap junction channels themselves to affect gating, assembly, and turnover, indirect action to liberate intracellular molecules may also produce rapid dynamic changes in spread of these factors throughout the coupled networks.

Cysteine residues in the cytoplasmic carboxy terminus of connexins dictate gap junction plaque stability

Cysteine residues within the cytoplasmic carboxyl-terminus of gap junction–forming proteins are required to stabilize gap junction plaque organization. The stability of gap junction plaque organization can be modified. Gap junction stability may provide a stable supramolecular platform for modulation of gap junction functions.