Bradford, Barry

Brophy, Peter

Manson, Jean

Tuzi, Nadia L.

Transmissible spongiform encephalopathies (TSEs) such as scrapie are neurodegenerative diseases characterised by long incubation periods and spongiform vacuolar pathology in the central nervous system (CNS). The expression of prion protein (PrP) is a requirement for the establishment and development of TSE disease. The process of agent replication in the periphery and invading the CNS is termed neuroinvasion. The exact mechanism of transport of the infectious agent is still unknown, and the roles of various cell types involved in this process are still to be determined. The TSE neuroinvasive process is postulated to be dependent upon a continuous chain of PrP expressing tissue between the infection site and the CNS, with the most likely candidate being the peripheral nervous system (PNS). However, measured transport rates of infection do not match rates of fast or slow axonal transport suggesting some other mechanism of transport. Myelinating Schwann cells of the PNS express PrP when associated with PrP expressing nerve axons and are capable of serially propagating the agent in vitro.  Transgenic mice possessing a Schwann cell specific knockout of the PrP gene were produced using the Cre / loxP system to test the hypothesis that Schwann cells are involved in TSE neuroinvasion. Following Cre-mediated recombination in these mice a 90 % reduction in total PrP expression and an altered PrP glycoform pattern were observed in peripheral nervous tissue. These mice were infected with mouse-adapted scrapie agents ME7 and 139A via intracerebral, intraperitoneal and oral routes of infection. No differences in incubation period or central nervous system pathology were observed when compared with peripherally infected control genotype mice. These results suggest that PrP expression in Schwann cells is not required for TSE neuroinvasion. <br/

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     Edinburgh Research Explorer                                      PRP Expression in Schwann Cells is not Required forTransmissible Spongiform Encephalopathy NeuroinvasionCitation for published version:Bradford, B, Manson, J, Brophy, P & Tuzi, NL 2007, 'PRP Expression in Schwann Cells is not Required forTransmissible Spongiform Encephalopathy Neuroinvasion' Prion 2007, Edinburgh, United Kingdom,26/09/07 - 28/09/07, .Link:Link to publication record in Edinburgh Research ExplorerDocument Version:Early version, also known as pre-printGeneral rightsCopyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s)and / or other copyright owners and it is a condition of accessing these publications that users recognise andabide by the legal requirements associated with these rights.Take down policyThe University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorercontent complies with UK legislation. If you believe that the public display of this file breaches copyright pleasecontact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately andinvestigate your claim.Download date: 28. Apr. 20171. Introduction Transmissible spongiform encephalopathies (TSE) are transmissible neurodegenerative diseases characterised by long incubation periods and characteristic pathology which includes vacuolar spongiform changes, apoptotic neuronal loss and astrocytic proliferation in the central nervous system (CNS). A diagnostic feature of these invariably fatal diseases is the presence of a protease resistant isoform (PrPSc) of the host-encoded membrane glycoprotein PrP, of which the protease sensitive isoform (PrPc) is necessary for disease susceptibility. Schwann cells are known to express PrP when associated with co-expressing peripheral nervous system axons and have also been shown to replicate and propagate TSE agent in culture. PrP Expression in Schwann Cells is Not Required for Transmissible Spongiform Encephalopathy Neuroinvasion Barry Bradford1, Peter Brophy2, Jean Manson1 and Nadia L. Tuzi1 1Neuropathogenesis Unit, Roslin Institute, Edinburgh, UK. 2Centre for Neuroscience Research, University of Edinburgh, Edinburgh, UK. Neuropathogenesis unit: Roslin Institute Funding: MRC. e-mail: barry.bradford@bbsrc.ac.uk a.PrPfl/flP 0 -Cre  PrPfl/fl P 0 -Cre wild type140150160170180190Genotype groupIncubation period (Days)b.PrPfl/flP 0 -Cre  PrPfl/fl P 0 -Cre wild type130140150160170180Genotype groupIncubation period (Days)C.wild typeP 0 -Cre P 0 -Cre  PrPfl/flPrPfl/fl200250300350400450500Genotype groupIncubation period (Days)e.wild typeP 0 -Cre P 0 -Cre  PrPfl/flPrPfl/fl150200250300350400450500Genotype groupIncubation period (Days)f.PrPfl/flP 0 -Cre  PrPfl/fl P 0 -Cre wild type150200250300350400450500Genotype groupIncubation period (Days)d.wild typeP 0 -Cre P 0 -Cre  PrPfl/fl PrPfl/fl225250275300325350375Genotype groupIncubation period (Days)Figure 3. Box-and-whisker diagram showing incubation period distribution following transmission of mouse-adapted Scrapie strains 139A (a, c, e) and ME7 (b, d, f) via intracerebral (a, b), oral (c, d) and intraperitoneal (e, f) routes.  Animals were grouped following post-mortem genotype confirmation, ANOVA revealed no significant differences between P0-Cre PrPfl/fl and control groups: , statistical outliers; ,  group mean incubation period. 6. Conclusion Removal of PrP expression in Schwann cells has no effect on TSE neuroinvasion of the two strains studied. We can therefore discount Schwann cells as a viable therapeutic target during the long incubation period between infection and clinical presentation. This study has revealed some very interesting biochemical and cell biological properties of PrP. The results from this study question what role PrP and its glycosylation status may play in disease. These results also offer insights into possible roles for PrP in glial / axonal interaction and cell-cell signalling. 2. Objectives To test the hypothesis that Schwann cells are involved in TSE neuroinvasion, a line of transgenic mice were produced using Cre / LoxP technology in which PrP expression is removed from Schwann cells. We have infected these mice with TSE agents via various routes to determine the difference in incubation period and CNS pathology when compared to wild type mice. 3. Methods By breeding mice (P0-Cre) expressing Cre under the control of the peripheral myelin Protein Zero (MPZ or P0) promoter (gift from M. Laura Feltri, Milan) and mice possessing LoxP sites flanking the PrP gene (PrPfl/fl), we have generated mice expressing PrP normally, except where P0 is expressed, i.e. myelinating Schwann cells. Mice were challenged with TSE agents 139A and ME7 intracerebrally (i.c.), orally and intraperitoneally (i.p.). Mice were clinically scored for signs of disease and analysed for terminal pathology. Disease incubation period, vacuolation in specific brain areas and presence of Proteinase K resistant PrP were analyzed as indicators of TSE disease. 5. Discussion Characterisation of these mice show that PrP is effectively excised, resulting in a 90 % reduction in PrPc expression in axonal lengths of peripheral nerves. This finding is particularly interesting as no differences have been observed in disease susceptibility or ability to traffic the infectious agent to the CNS in these mice. No alteration was seen in either incubation period or end pathology via agents 139A and ME7 after i.c., oral and i.p. challenges.  These results suggest that TSE neuroinvasion may occur via peripheral neurons alone with no involvement of peripheral glia despite their contribution to overall PrP expression in peripheral neurons. Also peripheral PrP glycosylation is not required for neuroinvasion and has no impact on strain specific properties such as the final targeting of CNS vacuolation and deposition of PrPSc. 4. Results Figure 4. Vacuolar pathology was assessed following transmission of mouse-adapted Scrapie strains 139A (a, c, e) and ME7 (b, d, f) via intracerebral (a, b), oral (c, d) and intraperitoneal (e, f) routes.  Brains were scored  on a scale of 0—5 in nine grey matter areas and 0–3 in three white matter areas, animals were grouped following post-mortem confirmation of genotype and mean scores calculated to produce lesion profiles (error bars representing ±SEM). Brain scoring areas: 1, dorsal medulla; 2, cerebellar cortex; 3, superior colliculus; 4, hypothalamus; 5. medial thalamus; 6, hippocampus; 7, septum; 8, cerebral cortex; 9, forebrain cerebral cortex; 10, cerebellar white matter; 11, midbrain white matter; 12, cerebral peduncle. , P0-Cre; , PrPfl/fl; p, wild type; , P0-Cre PrPfl/fl. Figure 2. a, Western analysis of PrPc expression in brain and sciatic nerve of P0-Cre PrPfl/fl (lanes marked *) , P0-Cre, wild-type and PrPfl/fl mice as detected by monoclonal antibody 7A12. Approximate protein size markers in kilodaltons (kDa).  b, Densitometric analysis of PrPc expression via Kodak 1D™ software showing reduction in PrPc expression between sciatic nerve from wild type and P0-Cre PrPfl/fl compound transgenic mice. Figure 1. PrPc (white) detected by fluorescence immunocytochemistry using monoclonal antibodies 7A12 and 8H4 (gift from Man Sun Sy, Cleveland). Sciatic nerve cross-section samples: a, wild type; b, P0-Cre PrPfl/fl and c, PrP0/0. Yellow arrows highlight strong PrP staining in myelin ring structures in wild type mice, red and green arrows show residual axonal and periaxonal staining respectively following conditional knockout of PrP expression from myelinating Schwann cells in P0-Cre PrPfl/fl compound transgenic mice. Fluorescence detection level was gated using PrP knockout mice. a. b. c. 

PRP Expression in Schwann Cells is not Required for Transmissible Spongiform Encephalopathy Neuroinvasion

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