Isolation of Terminal Schwann Cells by Fluorescence-Activated Cell Sorting
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abstract
The vertebrate neuromuscular junction (NMJ), like all synapses throughout the nervous system, hasthree cellular components: the presynaptic cell (the motor neuron), the postsynaptic cell (the skeletal musclefiber) and the glial wrappings (the nonmyelinating terminal Schwann cells (tSCs) that cap the nerve terminal).At the molecular level, we know the least about tSCs. Nevertheless, evidence suggests that mammalian tSCsplay important roles in re-establishment of synaptic connections following nerve damage in adult animals,regulate synapse pruning in neonates, and may have key roles at early stages of the neuromuscular diseasesamyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). Compelling as they are none of thesestudies directly tested whether tSC are necessary for each of these processes and what mechanisms arepossibly involved because there is a lack of genetic and molecular tools specifically targeting mammalian tSCs.This scarcity of tools is due to the paucity of proven tSC-specific markers. This R21 application proposes aninnovative method for isolating tSCs using fluorescence-activated cell sorting (FACS) that will allowidentification of tSC-specific markers at an unprecedented larger scale. The approach is based on comparingthe RNA-Seq-generated transcriptomes of myelinating and tSC-enriched cell pools derived by FACS frommuscle tissue in which fluorescent reporters of different colors are present in either tSCs or myelinating SCs.The expected outcome of this work is the identification of a largely complete set of those genes expressed intSCs but not in myelinating SCs. These markers then could be used in future experiments to selectivelymanipulate tSCs in vivo using molecular genetics and thus determine more definitely their contribution tosynaptic homeostasis in normal and pathological situations.
