Background & Aims

Neurofibromatosis 1 (NF1) is a genetic disorder affecting ~1/3000individuals that predisposes individuals to benign tumors in the peripheral nervous system (PNS), caused by the mutation in Nf1 gene, a negative regulator of the RAS/MAPK pathway. NF1 patients display multiple clinical manifestations including cutaneous and plexiform neurofibromas, café au lait spots, cognitive dysfunction, and learning disabilities. However, pain is the main debilitating feature that interferes with routine activities in patients with NF1. Emerging research has established the role of non-neuronal cells in nociception. Recent work from our lab has suggested that glial cell line-derived neurotrophic factor (GDNF) released from Schwann cells may play a role in the onset of pain due to NF1. The current study aims to determine if SC derived GDNF signals to its receptor GDNF family receptor-?1 (GFR?1) on neurons to regulate nociception in a preclinical model of NF1.

Methods

We performed behavioral assessments of mechanical sensitivity using a modified version of the mechanical conflict avoidance assay from mice with SC specific deletion of Nf1 (DhhCre;Nf1f/f) that were injected with an AAV9 containing a shRNA against GFR?1 into the sciatic nerves. Results were compared to control AAV injected mice. Data was correlated with gene expression using realtime PCR and immunohistochemistry on the DRGs for factors that have been linked to both tumor development and pain. Similarly, single unit electrophysiological recordings of sensory neurons using a novel ex vivo skin/nerve/dorsal root ganglion (DRG)/spinal cord preparation will be performed on the AAV injected mice. As macrophages have also been reported to play an important role in the onset of pain, and can release GDNF, the depletion of macrophages by systemic delivery of clodronate liposomes (i.v.) was performed prior to behavioral analyses to determine the role of other cells in onset of pain due to NF1.

Results

Nf1 deletion in SCs prior to tumor formation at 4-5 months of age produced mechanical hypersensitivity using choice-based behavioral assessments (mechanical conflict avoidance). This correlated with the sensitization of myelinated A-fiber nociceptors and unmyelinated polymodal C-fibers (CPM) to mechanical stimuli compared to controls using ex vivo recording. The mechanical hypersensitivity in the DhhCreNf1f/f mice was reduced to control levels after injection with AAV9-m-GFR?1-shRNA. The depletion of macrophages present in the nerves and DRGs of DhhCre; Nf1f/f mice by clodronate, however, did not alter the mechanical hypersensitivity observed in the NF1 mouse model when compared to controls.

Conclusions

The results from the current study showed that knocking down GFR?1 in sensory neurons reduces the mechanical hypersensitivity in the DhhCreNf1f/f mice, whereas depletion of macrophages had no direct effect on the observed mechanical hypersensitivity in the preclinical model of NF1. Together this indicates that GDNF-GFR?1 signaling from SC to neurons is a major contributor to pain in this tumor predisposition syndrome and targeting this pathway may be a novel cell-specific treatment strategy to ameliorate pain in NF1 patients.

References

NA

Presenting Author

Namrata G.R. Raut

Poster Authors

Namrata Raut

PhD

Cincinnati Children's Hospital Medical Center

Lead Author

E-mail

Topics

  • Models: Chronic Pain - Neuropathic