Terminal / perisynaptic Schwann cells (TPSCs) are a subtype of perisynaptic glial cell, located in the peripheral NS (PNS), that regulate the function, maintenance and regeneration of neuromuscular synapses. Recently, a role for TPSCs in regulating the early synaptic degeneration that occurs in animal models of motor neuron disease such as amyotrophic lateral sclerosis (ALS) has been demonstrated. To date, no study has selectively manipulated mouse TPSCs to examine their role in development and disease. In large part, this is due to the absence of a TPSC-specific marker that could be used to conduct genetic studies of these cells. Here, in order to selectively manipulate these cells in various contexts, we propose to create inducible Cre-driving mice under the promoter of KCNJ10. Our rationale for these studies is derived from preliminary data collected from ?first- generation? cell-specific genomic profiling of TPSCs. In these early studies, mice expressing epitope-tagged ribosomes (?RiboTag? mice) were crossed to Wnt1-Cre mice that drive expression of transgenes in TPSCs and nerve-associated axonal Schwann cells (ASCs). Subsequently, RNA from a sub-dissected region of the endplate region of diaphragm muscle that is enriched with TPSCs, as well as the phrenic motor nerve that contains only ASCs, was immunopurified, eluted, amplified and subjected to transcriptomic sequencing (RNA-Seq). We were able to identify and confirm several TPSC-specific genes, one of which encodes the inwardly rectifying potassium channel, Kir4.1, encoded by the KCNJ10 gene. Two issues remained after these early studies. One, despite the fact that Rosa26 knockin mice express a strong promoter designed to maintain strong expression of transgenes after recombination, Wnt1-Cre mice fail to drive reporter gene expression past early postnatal periods. This makes the use of the Wnt1-RiboTag approach infeasible to study the role of TPSCs in adults. Second, because Wnt1-Cre or Sox10-iCreERT2 mice drive expression in all Schwann cells, the best that can be achieved with micro-dissection of endplate muscle regions is an enrichment of TPSCs. Therefore, in this ?second generation? of TPSC genomic profiling, we propose to refine and therefore amplify their genomic signature by creating BAC transgenic mice expressing inducible Cre recombinase under the regulatory control of KCNJ10. We also plan to employ these mice to isolate the transcriptome of TPSCs at early stages of synaptic degeneration in young adult mutant ALS mice. Therefore, the proposed studies, if accomplished, provide a powerful tool to manipulate this important but often neglected perisynaptic glial cell.
The proposed research is relevant to public health because it proposes to create a tool to selectively manipulate terminal / perisynaptic Schwann cells, which regulate synaptic function and innervation in many peripheral neurodegenerative diseases and disorders. In Aim 2 of this proposal, we plan to isolate the transcriptome of these perisynaptic glial cells in early, pre- symptomatic stages of motor neuron disease. These studies may therefore provide novel targets for the treatment of this and other diseases of the neuromuscular junction.
