Isolation of Terminal Schwann Cells by Fluorescence-Activated Cell Sorting

项目总结

PROJECT SUMMARY The vertebrate neuromuscular junction (NMJ), like all synapses throughout the nervous system, has three cellular components: the presynaptic cell (the motor neuron), the postsynaptic cell (the skeletal muscle fiber) 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 tSCs play 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 diseases amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). Compelling as they are none of these studies directly tested whether tSC are necessary for each of these processes and what mechanisms are possibly 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 an innovative method for isolating tSCs using fluorescence-activated cell sorting (FACS) that will allow identification of tSC-specific markers at an unprecedented larger scale. The approach is based on comparing the RNA-Seq-generated transcriptomes of myelinating and tSC-enriched cell pools derived by FACS from muscle 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 in tSCs but not in myelinating SCs. These markers then could be used in future experiments to selectively manipulate tSCs in vivo using molecular genetics and thus determine more definitely their contribution to synaptic homeostasis in normal and pathological situations.