Glial cells must polarize on multiple axes. For example astrocytes, radial glia, myelin-forming glia or terminal Schwann cells engage in multiple cell-cell and cell-matrix interactions (i.e., with neurons, endothelial cells, muscle and basal lamina). This complex cytoarchitecture is crucial for glial function, but is also spatially inaccessible. As a result it is difficult to isolate specialized subcellular compartments for biochemical studies. Thus a major obstacle to the study of glia is the inaccessibility of molecular events occurring in relevant subcellular compartments. We have adapted a system, normally used to isolate polarized cell protrusion formed in response to soluble stimuli, to neuronal-Schwann cell interactions. We introduced the innovation of using neuronal cell membranes as stimulus, instead of soluble or extracellular matrix molecules, to mimic cell-cell interactions in glial cells. To this end we placed Schwann cells on a modified Boyden chamber with microporous filters, and exposed them to neuronal cell membranes in the bottom chamber. This causes Schwann cells polarization and extension of lamellipodia-like pseudopodia. Pseudopodia and cell bodies can then be physically separated and their contents compared. We performed proteomic and western blot analysis on these pseudopods, and found known molecules located at sites of axo-glial interactions, validating the system. We now propose to use this system to identify novel players in axo-glial interactions, using large sensory neurons and after addition of a second polarizing cue (extracellular matrix) to the Schwann cells. Next we will ask if the system can be used to study interactions between neurons and other glia, namely oligodendrocytes and astrocytes. The system can be adapted to multiple wild-type or mutant glia-cell interactions, to probe their role on protein or RNA polarization, formation of specific molecular complex or protein modification. These phenomena are relevant to physiological and pathological glial cell functions. This transformative resource could overcome the difficulty to study important glial undertakings at specialized cell junctions. !
We are developing an innovative system to study glial cell interactions and function in a culture dish. This system can be used to study the function of specialized domains of glial cells, which are important for normal brain, and peripheral nerve function and several neurological diseases.
|Nouri-Nigjeh, Eslam; Sukumaran, Siddharth; Tu, Chengjian et al. (2014) Highly multiplexed and reproducible ion-current-based strategy for large-scale quantitative proteomics and the application to protein expression dynamics induced by methylprednisolone in 60 rats. Anal Chem 86:8149-57|
|An, Bo; Zhang, Ming; Qu, Jun (2014) Toward sensitive and accurate analysis of antibody biotherapeutics by liquid chromatography coupled with mass spectrometry. Drug Metab Dispos 42:1858-66|
|Gui, Shanying; Gathiaka, Symon; Li, Jun et al. (2014) A remodeled protein arginine methyltransferase 1 (PRMT1) generates symmetric dimethylarginine. J Biol Chem 289:9320-7|
|Doddrell, Robin D S; Dun, Xin-Peng; Shivane, Aditya et al. (2013) Loss of SOX10 function contributes to the phenotype of human Merlin-null schwannoma cells. Brain 136:549-63|
|D'Antonio, Maurizio; Musner, Nicolo; Scapin, Cristina et al. (2013) Resetting translational homeostasis restores myelination in Charcot-Marie-Tooth disease type 1B mice. J Exp Med 210:821-38|
|Orita, Sumihisa; Henry, Kenneth; Mantuano, Elisabetta et al. (2013) Schwann cell LRP1 regulates remak bundle ultrastructure and axonal interactions to prevent neuropathic pain. J Neurosci 33:5590-602|
|Welser-Alves, Jennifer V; Boroujerdi, Amin; Tigges, Ulrich et al. (2013) Endothelial ?4 integrin is predominantly expressed in arterioles, where it promotes vascular remodeling in the hypoxic brain. Arterioscler Thromb Vasc Biol 33:943-53|