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.
|Ackerman, Sarah D; Luo, Rong; Poitelon, Yannick et al. (2018) GPR56/ADGRG1 regulates development and maintenance of peripheral myelin. J Exp Med 215:941-961|
|Poitelon, Yannick; Feltri, M Laura (2018) The Pseudopod System for Axon-Glia Interactions: Stimulation and Isolation of Schwann Cell Protrusions that Form in Response to Axonal Membranes. Methods Mol Biol 1739:233-253|
|Feltri, M Laura; Poitelon, Yannick; Previtali, Stefano Carlo (2016) How Schwann Cells Sort Axons: New Concepts. Neuroscientist 22:252-65|
|Poitelon, Y; Bogni, S; Matafora, V et al. (2015) Spatial mapping of juxtacrine axo-glial interactions identifies novel molecules in peripheral myelination. Nat Commun 6:8303|
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|Monk, Kelly R; Feltri, M Laura; Taveggia, Carla (2015) New insights on Schwann cell development. Glia 63:1376-93|
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