Neurochemical characterization of peripheral nerves infected with the scrapie agent during early neuroinvasion

Lindsay M. Fry¹, Huijun Yan², and David A. Schneider¹ . College of Veterinary Medicine^1,2, Veterinary Microbiology and Pathology², USDA ARS-ADRU², Washington State University

Scrapie is a fatal neurodegenerative disease of sheep and goats.  In natural disease, infectious prion protein (PrP^Sc) enters the body via the gastrointestinal tract and first replicates within ileal Peyer’s patches. Later in disease, the scrapie agent invades peripheral nerves from which it can disseminate to the central nervous system, accumulate and cause neurodegeneration, overt clinical disease, and death.  The site of peripheral neuroinvasion is not well understood though enteric and sympathetic nerve endings within the gastrointestinal tract are considered likely sites.  The neurons first infected with PrP^Sc are likely those that serve as the point of entry for PrP^Sc into the nervous system.  The goal of this study was to determine the neurochemical phenotype of the first neurons infected with the scrapie agent during early neuroinvasion.

Neurochemical phenotyping of peripheral neurons of interest was accomplished using immunohistochemical labeling of thin preparations of celiac ganglion and ileum harvested from two 7-month old (VVQQ) twin lambs inoculated with scrapie-positive placental cotyledons at birth.  In this, primary antibodies recognizing PrP^Sc and selected neurochemicals (neuronal NOS, Calbindin, Somatostatin, vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP), and tyrosine hydroxylase) were applied and visualized via application of appropriate secondary antibodies.

PrP^Sc was not detected in the brainstem or spinal cord of either lamb. Approximately 1.2% of neurons in the celiacomesenteric ganglion of one lamb were infected with PrP^Sc; PrP^Sc infection of the celiacomesenteric ganglion was not observed in the other lamb.  Approximately 12.4% and 14.6% of enteric neurons were infected in the two lambs, with the myenteric and submucosal plexuses equally affected.  These observations confirm that these lambs were in the initial stage of peripheral neural invasion. These results are consistent with the hypothesis that enteric neurons may serve as the initial portal of peripheral neuroinvasion from which central dissemination may occur via synaptic contact of autonomic nerve endings with infected enteric neurons.  VIP and somatostatin expressing neurons comprised the majority of PrP^Sc infected enteric neurons (36-48% and 28-31%, respectively).  These results suggest that the first neurons to become infected are enteric neurons that express VIP or somatostatin.  Studies are now underway to confirm these preliminary findings and to identify the probable sites of enteric PrP^Sc neuroinvasion by determining the projections of these VIP- and somatostatin-expressing enteric neurons.