Section on Cell Biology and Signal Transduction

We study the cellular mechanisms of calcium signaling by glial cells in the nervous system. Glial cells and neurons are in intimate communication with each other during central nervous system (CNS) development and normal brain function. Glial cells monitor and respond to neural activity by conditioning the extracellular milieu, signaling within glial cell networks, and sending signals back to neurons. In the brain, such signaling takes the form of propagated Ca²⁺ waves that spread over long distances in response to synaptic activity. Similarly, the myelinating glial cells (oligodendrocytes and Schwann cells) receive signals from the axon, with such signals essential for maintenance of the myelin sheath. One of our objectives is to understand the processes that support the temporal and spatial characteristics of Ca²⁺ signals within cells and between cells. A second objective is to understand the precise nature of glial cell signals in response to neuronal activity and the consequence of such signals for CNS function.

Ca²⁺-signaling microdomains

Holtzclaw, Russell

We have previously shown that Ca²⁺ wave propagation occurs through a series of Ca²⁺-signaling microdomains organized along glial cell processes. These microdomains act as highly specialized Ca²⁺ release sites that produce discretely localized spurts of cytoplasmic Ca²⁺ increases. In this way, wave propagation occurs by a fire-diffuse-fire cascade whereby the signal leaps along in a saltatory fashion. The specialized signaling microdomains are located 5 to 7 microns apart and are characterized by high-density patches of endoplasmic reticulum (ER) proteins involved in Ca²⁺ signaling, such as the inositol 1,4,5-trisphosphate receptors (IP₃Rs), sarco-endoplasmic reticulum calcium pumps, calreticulin, and at least one mitochondrion in close association (Haak et al., J Neurochem 2002;80:405; Holtzclaw et al., Glia 2002;39:69). In addition to supporting long-distance wave propagation, the specialized sites provide for locally discrete Ca²⁺ signals that last for only very brief periods. Complete knowledge of the proteins that make up the signaling microdomain and the mechanisms by which they support such highly organized Ca²⁺ release remains elusive.

Molecular characterization of signaling rafts

Weerth, Holtzclaw; in collaboration with Yergey

We investigate the molecular organization of Ca²⁺-signaling microdomains and describe their functional regulation in detail. Our overall aim is to test the hypothesis that, in the specialized microdomains of Ca²⁺ release, several macromolecular protein complexes are physically brought together, at times in different membrane systems, and interact to generate large spatially restricted Ca²⁺ signals. We have focused on glial cell processes, particularly cells of oligodendrocyte lineage and astrocytes, and propose to undertake a molecular characterization of these specialized Ca²⁺ release sites to determine the interacting protein assemblies that orchestrate the release process.

Our specific aim is to isolate, from glial cell membranes derived from mouse brains, intact oligomeric protein assemblies that make up specialized microdomains of Ca²⁺ signaling. We prepared cell-specific membranes from brain homogenates by using an immuno-affinity panning. We then used antibodies against cell-surface proteins to immuno-pan the homogenate in order to obtain subfractions containing membranes enriched in astrocyte membranes or oligodendrocyte progenitor (OP) cell membranes. We used Ran-2, an astrocytic surface antigen, or NG-2, an OP-cell antigen, for this purpose. We solubilized isolated membranes in detergent solution and used them either for immunoprecipitation or for separation of lipid raft–like structures by using isotonic density gradients.

We fractionated the gradients and tested each fraction in Western blots for protein content, using caveolin-1 as the marker for raft-like membranes and probing, with specific antibodies for a number of proteins involved in Ca²⁺ signaling. The proteins included surface membrane receptors M1 and P2Y1; surface membrane ion channel TrpC1; heterotrimeric G-protein isoform Gq; the scaffolding protein Homer; and endoplasmic reticulum membrane proteins IP3R2 and RyR3. The analysis showed that most of the proteins involved in Ca²⁺ signaling separated into low-density regions of the gradient, which also contained caveolin-1, suggesting that many signaling proteins exist in oligomeric complexes in cholesterol-rich and raft-like lipid domains.

In another experiment, we used solubilized cell-specific membranes as starting material and immunoprecipitated them with specific antibodies against IP3R2, Homer-1, and TrpC1 to determine the proteins constituting the oligomeric complexes. With the same three antibodies, we analyzed immunoprecipitated proteins again in Western blots. The experiment showed that IP3R2 antibodies specifically immunoprecipitated both TrpC1 and Homer-1. Similarly, anti-TrpC1 antibodoes precipitated Homer-1 and IP3R2 and anti-Homer-1 antibodies pulled down TrpC1 and IP3R2, suggesting that all three proteins were present in the oligomeric complexes. We separated the immunoprecipitated protein mixture on PAGE and stained gels with Coomasie brilliant blue. We then further analyzed several visualized protein bands by using high-resolution proteomic techniques for protein identification.

We carried out proteomic analysis in collaboration with Alfred Yergey. With protein bands excised and digested with trypsin, we used MALDI-TOF-TOF (matrix-assisted laser desorption/ionization tandem time-of-flight) mass spectrometry to analyze peptide pools in the tryptic digests. We compared peptide mass data with protein databases for positive hits. In an experiment currently in progress, we are also using tandem liquid chromatography–tandem mass spectrometry (LC-MS/MS for peptide mass determination and database comparison. We plan to select interesting subsets of proteins to investigate cellular localization and functional importance, followed by molecular identification. We now focus on myelinating glia in central and peripheral nerves as well as on astrocytic processes in situ. Initial experiments will involve immunocytochemical analysis of protein distribution in the context of the structural arrangement of glial cell membranes in the neuronal circuits and myelinated nerves.

Ca²⁺ signaling between axons and myelinating glia

Atkin; in collaboration with Ellisman

In a new project, we are investigating aspects of signaling between axons and their myelinating glia (Schwann cells and oligodendrocytes) in the peripheral and central nervous systems. Initially, we focused on characterizing the distribution of Ca²⁺-signaling proteins in the axoglial apparatus of the nodes of Ranvier. Serving as an electrical insulator, the myelin sheath reduces current flow across the axonal membrane in the internode. It also reduces capacitance and increases resistance, thereby facilitating saltatory conduction. Highly specialized extensions of myelinating glial cell (oligodendrocytes in the central nervous system and Schwann cells in the peripheral nervous system) membranes, intimately associated with axons, form myelin.

Beyond the concept of saltatory conduction, several studies support the view that the axon/myelin/glial cell ensemble, or “nodal complex,” operates in an integrated manner during conduction of the nerve impulse. Numerous anatomical and physiological studies over the last two decades have suggested that an intricate set of signals exist between the components of the nodal complex. Details of such signaling between axons and the myelinating glia are still unknown.

We initiated a study aimed at describing the distribution of proteins involved in Ca²⁺ signaling in the axoglial apparatus. We believed that the analysis would point us to spatial discreteness, if any, in the signaling between axons and Schwann cells. Our initial hypothesis posited that signaling is localized to specific contact sites between the two cell types where the insulating layer of compact myelin gives way to non-compact myelin and Schwann cell membrane. We selected antibodies against proteins involved in Ca²⁺ signaling (e.g., IP3Rs and RyRs) as well as structural proteins unique to different regions of the node and investigated their discrete localization by using immunocytochemistry followed by high-resolution fluorescence microscopy.

We performed the analysis in an isolated, teased sciatic nerve preparation, both fixed and unfixed. The distribution of myelin basic protein (MBP) and S100beta showed the previously described intricate architecture of the nodes of Ranvier and highlighted the intimate relationship between the axon and the myelinating glia. We focused on the arrangement of proteins in the axoglial apparatus and selected specific proteins uniquely expressed to the node: (Na_v1.2), paranode (MAG, connexin 32), the juxtaparanode (Kv 1.2), and the Schwann cell cytoplasm (S100beta). Immunohistochemical analysis using antibodies against these selected proteins provided us with the anatomical arrangements of the two cell types in the nodes of Ranvier. We examined the distribution of proteins involved in Ca²⁺ signaling within the architecture of the myelinated axon, using specific antibodies against IP3Rs, RyRs, P2Y₁, M₁, and G_q. In future experiments, we plan to investigate the distribution of the signaling proteins at higher resolution by using electronmicroscopic immunocytochemical techniques. We conducted the experiments collaboratively with Mark Ellisman. We plan to focus on co-localization of specific proteins in discrete regions of the Schwann cells.

In situ imaging of Schwann cell calcium signals

Atkin; in collaboration with Pickel

During the past year, we began to measure directly Schwann cell Ca²⁺ signals associated with action potential traffic along axons. To this end, we developed a transgenic mouse that would express a fluorescent Ca²⁺ indicator in a specific manner in Schwann cells. We used the S-100b promoter to target specifically a mutant chameleon protein, YC3.60, to Schwann cells in the peripheral nerves and to astrocytes in the brain. We obtained a YC3.60 construct from Atsushi Miyawaki at RIKEN Brain Science Institute, Tokyo, Japan, and engineered a plasmid construct containing the S100b promoter in tandem. We produced two such plasmid constructs: S100-YC3.60.6 and S100-YC3.60.9. Transfection of C6 glioma cell lines and HEK 293 cell lines showed that YC3.60 was expressed only by the glioma line, which expresses S100b and not in HEK293 cells. In collaboration with James Pickel, we used the plasmid DNA to generate transgenic mouse founders. Using PCR genotyping and the level of specific YC3.60 expression in astrocytes in the brain as a criterion, we found 11 founder mice. We are currently generating individual lines from these founders in order to obtain homozygous colonies.

We propose to use tissue from these animals and the animals themselves to investigate a number of issues regarding glial cell signaling. We will record action potential–dependent Ca²⁺ signals in Schwann cells in both isolated nerve trunks and intact animals by applying dual-photon confocal microscopy, which allows for the use of infrared illumination and deep tissue imaging such that signals can be recorded many hundreds of micrometers deep within tissue. We propose to image nerve trunks both in vitro and in vivo under dual-photon microscopy during electrical stimulation. The new transgenic mice express YC3.60 in all astrocytes within the brain, thus providing the opportunity to monitor astrocytic Ca²⁺ signals discretely in the brain of living animals as well as in isolated tissue and cells.

Kárai LJ, Russell JT, Iadarola MJ, Oláh Z. Vanilloid receptor 1 regulates multiple calcium compartments and contributes to Ca²⁺-induced Ca²⁺-release in sensory neurons. J Biol Chem 2004;279:16377-16387.

Voulalas PJ, Holtzclaw L, Wolstenholme J, Russell JT, Hyman SE. Metabotropic receptors and dopamine receptors cooperate to enhance extracellular signal-regulated kinase phosphorylation in striatal neurons. J Neurosci 2005;25:3763-3773.

Yang F, He XP, Russell J, Lu B. Ca²⁺ influx–independent synaptic potentiation mediated by mitochondrial Na⁺-Ca²⁺ exchanger and protein kinase C. J Cell Biol 2003;163:511-523.

CollaboratorS

Mark Ellisman, PhD, University of California San Diego, La Jolla, CA

James Pickel, PhD, Laboratory of Genetics, Transgenic Core Facility, NIMH, Bethesda, MD

Alfred Yergey, PhD, Laboratory of Cellular and Molecular Biophysics, NICHD, Bethesda, MD

For further information, contact james@helix.nih.gov.