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P&F Grant Awards

Year 8

Grant # 17

Molecular and Genetic Analysis of Terminal Schwann Cell Function in Homeostasis and Injury.

PI: Alison Snyder-Warwick, MD


Specific Aims

Functional outcomes following peripheral nerve injuries diminish with the time required for regenerating axons to reach their muscle target. Terminal Schwann cells (tSCs) are supportive glial cells located at the muscle target that are relatively understudied, but are attractive targets for protecting denervated muscle. Over the past 2.5 years, my laboratory has optimized techniques for tSC investigation. This proposal focuses on tSCs and will describe their unique genetic profiles, their impact on homeostasis and neuromuscular junction (NMJ) reinnervation after injury, and the mechanisms by which they respond to motor nerve injury.

Schwann cells (SCs), the principal glial cells of the peripheral nervous system (PNS), are composed of two main subtypes: myelinating and non-myelinating. tSCs are non-myelinating SCs that cover motor nerve terminals and contribute to formation, maintenance, and regeneration of the synapse [1-8]. Relatively little, however, is known about tSC signaling during these events. The goals of this proposal are to: 1) identify unique genetic markers of tSCs to improve tools for investigation, 2) determine the impact of tSCs on (a) synaptic transmission in homeostasis and (b) NMJ reinnervation after motor nerve injury, and 3) determine the mechanisms by which tSCs contribute to NMJ reinnervation after injury.

Specific Aim #1. To identify genes uniquely expressed in tSCs. Hypothesis: tSCs have unique genetic expression profiles that differ from other SCs. There are no known genetic markers that are unique to tSCs. We will employ microarray analysis of tSCs isolated from S100-GFP mice, whose SCs fluoresce, using: 1) a novel component dissection technique and subtraction analysis and 2) cell culture. Candidate genes will be validated in vivo with in situ hybridization and RT-PCR, and protein expression will be assessed with immunofluorescence. Our ultimate goal is to build the tools to generate a transgenic mouse line with Cre-mediated recombination in tSCs to facilitate further tSC investigation.

Specific Aim #2.To determine: a) the functional consequences of tSC loss in homeostasis and b) the requirement for tSC contributions to NMJ reinnervation following motor nerve injury. Hypotheses: (a) Efficiency of synaptic transmission decreases after tSC ablation resulting in reduced muscle force. (b) NMJ reinnervation is compromised after tSC ablation due to loss of axon sprouting, guidance, and trophic support, resulting in diminished or absent muscle force. tSCs interact with neurotransmitters in homeostasis [6], implicating importance for synaptic maintenance. Following motor nerve injury, tSC processes extend, induce nerve sprouting, and guide regenerating axons to adjacent NMJs, suggesting importance for reinnervation [1, 2, 9, 10]. We will perform immune-mediated tSC ablation in homeostasis and at multiple time points following nerve transection and repair. The effects of tSC ablation will be assessed functionally via muscle force testing and morphologically with confocal microscopy.

Specific Aim #3. To determine the mechanisms active during the tSC response after motor nerve injury. Hypotheses: The tSC response to motor nerve injury mimics myelinating SC development and response to injury with p38 MAPK activation. Myelinating SC elongation during development is mediated by the p38 MAPK signaling [11], and multiple MAPK pathways modulate myelinating SC dedifferentiation to a more immature state in response to injury [12-14]. Activation of ErbB2 mimics tSC response to injury [15]. We hypothesize that tSC process elongation after motor nerve injury is mediated via p38 MAPK activation. We will perform immunohistochemical and functional analyses of the MAPK pathways following nerve transection in different transgenic mouse models that facilitate in vivo evaluation of this pathway.

Twenty million Americans suffer from peripheral nerve injury [16], and management of peripheral nerve pathology remains temporally limited by the ability to successfully reinnervate the target muscle. With their location at the nerve-muscle interface and functional plasticity, tSCs provide an opportunity to decipher events at the muscle target. Knowledge of tSC biology may provide innovative strategies to manipulate their molecular signature to protect the muscle target, lengthen the window during which reinnervation may occur, and improve motor function. The proposed experiments unite important questions from glial biology and challenges in clinical management of peripheral nerve injuries to establish an innovative and translationally important investigative focus.