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Temporal Resolution of Misfolded Prion Protein Transport, Accumulation, Glial Activation, and Neuronal Death in the Retinas of Mice Inoculated with Scrapie

2016; Elsevier BV; Volume: 186; Issue: 9 Linguagem: Inglês

10.1016/j.ajpath.2016.05.018

1525-2191

M. Heather West Greenlee, Melissa Lind, Robyn Kokemuller, Najiba Mammadova, Naveen Kondru, Sireesha Manne, Jodi D. Smith, Anumantha G. Kanthasamy, Justin J. Greenlee,

Neurological diseases and metabolism

Currently, there is a lack of pathological landmarks to describe the progression of prion disease in vivo. Our goal was to use an experimental model to determine the temporal relationship between the transport of misfolded prion protein (PrPSc) from the brain to the retina, the accumulation of PrPSc in the retina, the response of the surrounding retinal tissue, and loss of neurons. Retinal samples from mice inoculated with RML scrapie were collected at 30, 60, 90, 105, and 120 days post inoculation (dpi) or at the onset of clinical signs of disease (153 dpi). Retinal homogenates were tested for prion seeding activity. Antibody staining was used to assess accumulation of PrPSc and the resulting response of retinal tissue. Loss of photoreceptors was used as a measure of neuronal death. PrPSc seeding activity was first detected in all samples at 60 dpi. Accumulation of PrPSc and coincident activation of retinal glia were first detected at 90 dpi. Activation of microglia was first detected at 105 dpi, but neuronal death was not detectable until 120 dpi. Our results demonstrate that by using the retina we can resolve the temporal separation between several key events in the pathogenesis of prion disease. Currently, there is a lack of pathological landmarks to describe the progression of prion disease in vivo. Our goal was to use an experimental model to determine the temporal relationship between the transport of misfolded prion protein (PrPSc) from the brain to the retina, the accumulation of PrPSc in the retina, the response of the surrounding retinal tissue, and loss of neurons. Retinal samples from mice inoculated with RML scrapie were collected at 30, 60, 90, 105, and 120 days post inoculation (dpi) or at the onset of clinical signs of disease (153 dpi). Retinal homogenates were tested for prion seeding activity. Antibody staining was used to assess accumulation of PrPSc and the resulting response of retinal tissue. Loss of photoreceptors was used as a measure of neuronal death. PrPSc seeding activity was first detected in all samples at 60 dpi. Accumulation of PrPSc and coincident activation of retinal glia were first detected at 90 dpi. Activation of microglia was first detected at 105 dpi, but neuronal death was not detectable until 120 dpi. Our results demonstrate that by using the retina we can resolve the temporal separation between several key events in the pathogenesis of prion disease. Transmissible spongiform encephalopathies (TSEs) are a family of diseases caused by the accumulation of misfolded prion protein (PrPSc).1Prusiner S.B. Molecular biology of prion diseases.Science. 1991; 252: 1515-1522Crossref PubMed Scopus (1745) Google Scholar During progression of TSEs, like many protein misfolding disorders, transport of misfolded protein from one central nervous system (CNS) structure seeds protein misfolding and accumulation in another.2Shearin H. Bessen R.A. Axonal and transynaptic spread of prions.J Virol. 2014; 88: 8640-8655Crossref PubMed Scopus (18) Google Scholar The details underlying this series of events that begins with the arrival of misfolded protein in a CNS structure and ends with neuronal death in that structure are not well understood. Seeding the brain with an inoculum of misfolded prion protein induces TSEs; thus, these diseases provide a unique opportunity to study the transport of PrPSc from one CNS structure to another. Currently, there is no treatment for TSEs. Although in silico and in vitro approaches have been effective at identifying compounds with therapeutic potential,3Cashman N.R. Caughey B. Prion diseases: close to effective therapy?.Nat Rev Drug Discov. 2004; 3: 874-884Crossref PubMed Scopus (106) Google Scholar, 4Zhou M. Ottenberg G. Sferrazza G.F. Hubbs C. Fallahi M. Rumbaugh G. Brantley A.F. Lasmezas C.I. 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Optical coherence tomography in Alzheimer's disease: a meta-analysis.PLoS One. 2015; 10: e0134750Crossref Scopus (139) Google Scholar Parkinson disease,10Bodis-Wollner I. Kozlowski P.B. Glazman S. Miri S. Alpha-synuclein in the inner retina in parkinson disease.Ann Neurol. 2014; 75: 964-966Crossref PubMed Scopus (123) Google Scholar, 11Beach T.G. Carew J. Serrano G. Adler C.H. Shill H.A. Sue L.I. Sabbagh M.N. Akiyama H. Cuenca N. Arizona Parkinson's Disease Consortium: Phosphorylated alpha-synuclein-immunoreactive retinal neuronal elements in Parkinson's disease subjects.Neurosci Lett. 2014; 571: 34-38Crossref PubMed Scopus (85) Google Scholar, 12Chorostecki J. Seraji-Bozorgzad N. Shah A. Bao F. Bao G. George E. Gorden V. Caon C. Frohman E. Tariq Bhatti M. Khan O. Characterization of retinal architecture in Parkinson's disease.J Neurol Sci. 2015; 355: 44-48Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar and numerous TSEs, including scrapie in sheep,13Greenlee J.J. Hamir A.N. West Greenlee M.H. Abnormal prion accumulation associated with retinal pathology in experimentally inoculated scrapie-affected sheep.Vet Pathology. 2006; 43: 733-739Crossref PubMed Scopus (31) Google Scholar, 14Regnier A. Andreoletti O. Albaric O. Gruson D.C. Schelcher F. Toutain P.L. Clinical, electroretinographic and histomorphometric evaluation of the retina in sheep with natural scrapie.BMC Vet Res. 2011; 7: 25Crossref PubMed Scopus (10) Google Scholar chronic wasting disease in cervids,15Spraker T.R. O'Rourke K.I. Gidlewski T. Powers J.G. Greenlee J.J. Wild M.A. Detection of the abnormal isoform of the prion protein associated with chronic wasting disease in the optic pathways of the brain and retina of Rocky Mountain elk (Cervus elaphus nelsoni).Vet Pathol. 2010; 47: 536-546Crossref PubMed Scopus (17) Google Scholar, 16Keane D.P. Barr D.J. Bochsler P.N. Hall S.M. Gidlewski T. O'Rourke K.I. Spraker T.R. Samuel M.D. Chronic wasting disease in a Wisconsin white-tailed deer farm.J Vet Diagn Invest. 2008; 20: 698-703Crossref PubMed Scopus (68) Google Scholar, 17Hamir A.N. Greenlee J.J. Nicholson E.M. Kunkle R.A. Richt J.A. Miller J.M. Hall M. Experimental transmission of chronic wasting disease (CWD) from elk and white-tailed deer to fallow deer by intracerebral route: final report.Can J Vet Res. 2011; 75: 152-156PubMed Google Scholar bovine spongiform encephalopathy in cattle,18Greenlee J.J. Smith J.D. West Greenlee M.H. Nicholson E.M. Clinical and pathologic features of H-type bovine spongiform encephalopathy associated with E211K prion protein polymorphism.PLoS One. 2012; 7: e38678Crossref PubMed Scopus (28) Google Scholar, 19Greenlee M.H. Smith J.D. Platt E.M. Juarez J.R. Timms L.L. Greenlee J.J. Changes in retinal function and morphology are early clinical signs of disease in cattle with bovine spongiform encephalopathy.PLoS One. 2015; 10: e0119431Crossref Scopus (41) Google Scholar and Creutzfeldt-Jakob disease in humans.20Head M.W. Northcott V. Rennison K. Ritchie D. McCardle L. Bunn T.J. McLennan N.F. Ironside J.W. Tullo A.B. Bonshek R.E. Prion protein accumulation in eyes of patients with sporadic and variant Creutzfeldt-Jakob disease.Invest Ophthalmol Vis Sci. 2003; 44: 342-346Crossref PubMed Scopus (83) Google Scholar, 21Head M.W. Peden A.H. Yull H.M. Ritchie D.L. Bonshek R.E. Tullo A.B. Ironside J.W. Abnormal prion protein in the retina of the most commonly occurring subtype of sporadic Creutzfeldt-Jakob disease.Br J Ophthalmol. 2005; 89: 1131-1133Crossref PubMed Scopus (29) Google Scholar The retina and associated visual structures provide an excellent model to study the transport of misfolded proteins from one CNS structure to another.22Rahimi J. Milenkovic I. Kovacs G.G. Patterns of tau and alpha-synuclein pathology in the visual system.J Parkinsons Dis. 2015; 5: 333-340Crossref PubMed Scopus (9) Google Scholar Herein, we report the time course of transport of misfolded prion protein to the retina after inoculation into the brain in a mouse model of scrapie. We describe the temporal separation between the first detection of misfolded prion protein at 40% of the 153-day incubation period, the accumulation of PrPSc at 60% of the incubation period, the activation of microglia at 70% of the incubation period, and the loss of retinal neurons at 80% of the incubation period. Thus, we provide a well-described in vivo model that can be used to study the pathogenesis of disease and to assess the potential effectiveness of therapeutic approaches against the spread and accumulation of misfolded prion protein. The experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals.23Committee for the Update of the Guide for the Care and Use of Laboratory AnimalsNational Research CouncilGuide for the Care and Use of Laboratory Animals: Eighth Edition. National Academies Press, Washington, DC2011Crossref Google Scholar The protocol was approved by the Institutional Animal Care and Use Committee at the National Animal Disease Center (protocol number 3985). Sample size was determined statistically using preliminary data from clinically affected animals. C57Bl/6 mice, aged 6 to 8 weeks, were inoculated with a brain homogenate containing mouse-adapted Rocky Mountain Laboratories (RML) scrapie via intracranial injection into the right cerebral hemisphere at the level of the temporal bone. Tissue collection occurred at 30, 60, 90, 105, and 120 days post inoculation (dpi), along with another group composed of animals exhibiting clinical signs approximately 155 dpi. For each time point, there were two groups of age-matched negative control mice: a mock-inoculated group that was inoculated with brain homogenate from healthy mice and a noninoculated group that received no inoculation (five to six animals were included in each group). Globes were collected into Bouin's fixative for 24 hours and post-fixed in 10% formalin until processing. Tissues were then embedded in paraffin, divided into sections (4 μm thick), and affixed to slides for hematoxylin and eosin staining and immunolabeling. Recombinant prion protein (rPrP) used in RT-QuIC assay was expressed and purified using standard protocol from previous reports.24Wilham J.M. Orru C.D. Bessen R.A. Atarashi R. Sano K. Race B. Meade-White K.D. Taubner L.M. Timmes A. Caughey B. Rapid end-point quantitation of prion seeding activity with sensitivity comparable to bioassays.PLoS Pathog. 2010; 6: e1001217Crossref PubMed Scopus (318) Google Scholar, 25Atarashi R. Wilham J.M. Christensen L. Hughson A.G. Moore R.A. Johnson L.M. Onwubiko H.A. Priola S.A. Caughey B. Simplified ultrasensitive prion detection by recombinant PrP conversion with shaking.Nat Methods. 2008; 5: 211-212Crossref PubMed Scopus (234) Google Scholar Briefly, rPrP composed of the Syrian hamster residues 90 to 231 in pET vector with N-terminal histidine sequence was transformed in to Escherichia coli Rosetta2(DE3) cells. The cultures were grown under continuous selection with kanamycin and chloramphenicol in the Lysogeny broth media. The log phase cultures were scaled up with Overnight Express Autoinduction System 1 for protein induction. Later, the inclusion bodies were isolated from bacterial pellets using BugBuster Master Mix and were denatured in 8M guanidine hydrochloride. Later, the rPrP is bound to pre-equilibrated nickel-nitrilotriacetic acid superflow resin for 50 minutes. The beads were packed in Akta #XK26 column, and rPrP was purified using nickel-nitrilotriacetic acid affinity chromatography on a Bio-Rad Duo flow system in a refrigerated room. On column refolding was performed using gradient dilution of guanidine hydrochloride over 5 hours. Pure rPrP was eluded from the column using imidazole binding with increasing linear gradient. Chilled dialysis buffer was added to the peak elude fractions, and dialyzed overnight against three changes of chilled dialysis buffer (10 mmol/L NaPO4, pH 5.8) after initial filtration. Protein concentration was determined using the extension coefficient and filtering through 100-kDa cutoff filters before subjecting to RT-QuIC assay. The quality of rPrP was confirmed by NMR and CD spectral analyses. The RT-QuIC assay was performed using previously published protocols from published reports24Wilham J.M. Orru C.D. Bessen R.A. Atarashi R. Sano K. Race B. Meade-White K.D. Taubner L.M. Timmes A. Caughey B. Rapid end-point quantitation of prion seeding activity with sensitivity comparable to bioassays.PLoS Pathog. 2010; 6: e1001217Crossref PubMed Scopus (318) Google Scholar, 26Haley N.J. Hoover E.A. Chronic wasting disease of cervids: current knowledge and future perspectives.Annu Rev Anim Biosci. 2015; 3: 305-325Crossref Scopus (126) Google Scholar, 27Orru C.D. Groveman B.R. Hughson A.G. Zanusso G. Coulthart M.B. Caughey B. Rapid and sensitive RT-QuIC detection of human Creutzfeldt-Jakob disease using cerebrospinal fluid.MBio. 2015; 6 (e02451-14)Crossref PubMed Scopus (161) Google Scholar with slight modifications. The reaction mixtures consisted of final concentrations of 350 mmol/L NaCl, 0.1 mmol/L EDTA, 10 μmol/L thioflavin T, 0.1 mg/mL rPrP, and 0.002% SDS in 1× phosphate-buffered saline. The retina samples from time course RML scrapie-infected mice were washed twice and homogenized using repeated pipetting and sonication in a cup sonicator with two pulses of 30 seconds. The clarified supernatants were normalized to protein concentration using Bradford protein assay. The reactions consisted of 5 μg of protein from retina homogenates that were used as seed. We included mouse brain homogenates (0.5 μg) from clinical RML scrapie infected mice, mock brain homogenates, and blank (buffer) as controls. All of the samples were run using a blinded study design (N.K., S.M., A.K.). The plates were sealed with Nalgene Nunc plate sealer and subjected to RT-QuIC assay. The plates were incubated at 42°C in Cytation3 (Biotek, Winooski, VT) plate reader with alternate cycles of 1-minute shake (double orbital) and rest. All of the samples were run in quadruplicates, and samples were judged to be positive as reported previously.27Orru C.D. Groveman B.R. Hughson A.G. Zanusso G. Coulthart M.B. Caughey B. Rapid and sensitive RT-QuIC detection of human Creutzfeldt-Jakob disease using cerebrospinal fluid.MBio. 2015; 6 (e02451-14)Crossref PubMed Scopus (161) Google Scholar Average fluorescence readings of four replicates plus 10 times SD of negative controls were the criteria for determining the threshold. Recording of thioflavin T fluorescence (450 ± 15 nm excitation and 480 ± 10 nm emission) was noted every 30 minutes, and data analysis was performed using Biotek's Gen5 software version 2.07.17. Paraffin-embedded tissues were analyzed using immunohistochemistry. After deparaffinization and rehydration, tissue sections were autoclaved for 20 minutes in an antigen retrieval solution (DAKO Target Retrieval Solution; Dako Corp., Carpinteria, CA). Mouse anti-PrP (6C2; CVI-WUR, Lelystad, Neth) was diluted 1:2000. Rabbit anti-glial fibrillary acidic protein (GFAP) (Dako Corp.) was diluted at 1:10,000. Slides were incubated in primary antibodies 6C2 overnight at 4°C and GFAP for 24 hours at room temperature and 72 hours at 4°C. Immunoreactivity was developed using Envision + Dual Link System-horseradish peroxidase (Dako Corp.) with diaminobenzidene (Vector Labs, Peterborough, UK), and slides were counterstained with hematoxylin. Images were captured using a Nikon DS camera on a Nikon Eclipse 50i microscope. For immunofluorescence detection of CD68 immunoreactivity (1:100; Dako Corp.), slides were incubated with Cy3 conjugated AffiniPure secondary antibodies (1:300; Jackson ImmunoResearch, West Grove, PA) for 1.5 hours and counterstained with DAPI (1 μg/mL; Sigma-Aldrich, St. Louis, MO). Immunofluorescence images were captured using a commercial upright microscope system (Zeiss AxioPlan 2 Microscope Imaging System; Oberkochen, Germany). CD68 immunoreactivity was quantified using ImageJ analysis software version 1.49v (http://imagej.nih.gov/ij; NIH, Bethesda, MD), and was expressed as a percentage of the total image area (outer limiting membrane to inner limiting membrane of the central retina) using constant thresholds. Values at each time point were pooled and analyzed with one-way analysis of variance and Tukey's post hoc test using Prism 6 for Windows (Graph Pad Software, San Diego, CA). Thinning of the outer nuclear layer was measured by counts of cell bodies spanning the layer. For each cross section, three counts were made in the central retina and averaged into a single value for each animal. Data were analyzed with one-way analysis of variance using Dunnett's multiple comparison test. Seventeen C57Bl/6 mice were used to determine the incubation period of mouse-adapted (RML) scrapie. The average incubation period was 153 dpi, with a SD of 1.4 days. On the basis of a 153-day incubation period, tissues were assayed at 19.6% of total incubation (30 dpi), 39.2% of total incubation (60 dpi), 58.8% of incubation (90 dpi), 68.6% of incubation (105 dpi), 78.4% of incubation (120 dpi), and 100% of incubation period (clinical illness). The real-time quaking induced conversion (RT-QuIC) assay was used to determine the time course of misfolded prion protein transport from the brain to the retina. Seeding activity in the RT-QuIC assay was noted in all replicates (both biological and technical) of clinical, 120 dpi, 90 dpi, and 60 dpi retina samples but not in retinas from mock inoculated animals or from 30 dpi inoculated retina samples (Figure 1A). Seeding activity in retinal samples was comparable to the RML scrapie infected brain homogenate (Figure 1A). When data were expressed as amyloid formation rate [the inverse of the time for the thioflavin T fluorescence signals to cross the threshold (in hours)], it demonstrated that amyloid formation rate is proportional to the incubation period. That is, the longer the course of the disease, the greater the rate of amyloid formation (Figure 1B). The mean rate of amyloid formation from retina of 30, 60, 90, and 120 dpi and clinical were 0, 0.06, 0.12, 0.17, and 0.14 hours−1, respectively. Immunohistochemistry was used to assess PrPSc accumulation in retinal sections from mice at 30, 60, 90, 105, and 120 dpi and at the time clinical illness was observed (Figure 2). Mock-infected animals (collected at the same time points) were used as controls. There was no immunostaining for PrPSc in any of the control animals (Figure 2A). Immunostaining was first observed at 90 dpi (at approximately 60% of the total incubation period) in multifocal photoreceptor inner segments throughout the retinal sections of each mouse examined (Figure 2B). Fifteen days later, at 105 dpi, immunoreactivity in photoreceptor inner segments was more widespread, and PrPSc immunoreactivity also was observed in the outer plexiform (synaptic) layer (Figure 2C). At 120 dpi, PrPSc immunoreactivity was observed in the inner plexiform layer in addition to the photoreceptor layer and outer plexiform layer (Figure 2D). At the time of clinical disease (approximately 153 dpi), PrPSc immunoreactivity was observed in the inner and outer plexiform layers and in the photoreceptors that remained (Figure 2E) (although by this time point, most photoreceptors had been lost). Müller glia are radial glial cells that are derived from retinal progenitor cells.28Cepko C. Intrinsically different retinal progenitor cells produce specific types of progeny.Nat Rev Neurosci. 2014; 15: 615-627Crossref PubMed Scopus (211) Google Scholar Quiescent Müller glia are immunoreactive for GFAP in their end feet, which are in the optic fiber layer. Under conditions of retinal stress, activated Müller glia have GFAP immunoreactivity more widely distributed in their processes that span from the optic fiber layer to the outer limiting membrane.29Lewis G.P. Fisher S.K. Up-regulation of glial fibrillary acidic protein in response to retinal injury: its potential role in glial remodeling and a comparison to vimentin expression.Int Rev Cytol. 2003; 230: 263-290Crossref PubMed Scopus (302) Google Scholar Immunohistochemistry with an antibody against GFAP was used to assess activation of Müller glia in retinal sections from 30, 60, 90, 105, and 120 dpi and at the stage of clinical illness (Figure 3). Mock-inoculated animals collected at each time point were used as controls. In control animals, GFAP immunoreactivity was observed in the optic fiber layer (Figure 3A) that contains astrocytes and the end feet of Müller glia. There was not an appreciable difference between GFAP immunoreactivity in mock, 30 dpi, or 60 dpi (data not shown). However, GFAP immunoreactivity at 90 dpi was observed in processes of Müller glia that extended as far as the outer plexiform layer (Figure 3B). At 105 dpi, GFAP immunoreactivity was more robust, but still only observed between the optic fiber layer and the outer plexiform layer (Figure 3C). GFAP immunoreactivity at 120 dpi also was observed in the outer nuclear layer and reached the outer limiting membrane in some areas (Figure 3D). At the clinical stage of disease, there was severe loss of photoreceptors and Müller glia hypertrophy with GFAP immunoreactivity observed throughout the thickness of the retina (Figure 3E). An antibody against CD68 was used to assess activation of microglia in retinal sections from mice at dpi 30, 60, 90, 105, 120, and the clinical stage of disease in comparison to mock-inoculated control animals. Rare, multifocal CD68 immunoreactivity was observed in the outer plexiform layer of control retinal sections (Figure 4A). CD68 immunoreactivity in retinal sections from mice examined at 30 or 60 dpi (data not shown) or 90 dpi (Figure 4B) was not appreciably different from controls. However, at 105 dpi, CD68 immunoreactivity was more intense and widespread in the outer plexiform layer and also was observed multifocally in the inner plexiform layer and the optic fiber layer (Figure 4C). Immunoreactivity was further increased at 120 dpi (Figure 4D). In retinal sections from clinically ill mice, CD68 immunoreactivity was most intense in the optic fiber layer and the inner plexiform layer (Figure 4E). When CD68 immunoreactivity was quantified at each time point, it illustrated that the first significant increase in CD68 immunoreactivity compared to control animals was at the 105 dpi time point (P < 0.05) (Figure 4F) or at approximately 70% of incubation period. To determine the time course of photoreceptor loss during RML scrapie incubation, outer nuclear layer thickness (as measured by number of photoreceptor cell nuclei) was assessed at 30, 60, 90, 105, and 120 dpi and in the retinas from animals with clinical disease (Figure 5). In noninoculated control animals and mock-inoculated animals, the mean outer nuclear layer thickness was 11.05 cell nuclei (±0.81). The mean outer nuclear layer thickness was not appreciably different from controls at 30, 60, 90, or 105 dpi. At 120 dpi, however, the average outer nuclear layer thickness was significantly decreased to 8.94 (±1.00) cell nuclei (P < 0.01) and decreased to an average of 2.08 (±0.50) cell nuclei thick in retinas from animals with clinical disease. Thus, neuronal death in the form of significant photoreceptor loss (P < 0.01) was not detected in the retinas until approximately 80% of total incubation period. Our results demonstrate seeding activity by RT-QuIC in the retina at 60 dpi (approximately 40% of incubation period) in all samples assayed. Prion seeding activity in the retina was detected 30 days before the detection of PrPSc via immunohistochemistry. This is consistent with a report that compared RT-QuIC with immunohistochemistry and found RT-QuIC could detect prion seeding activity in tissues that were negative by immunohistochemistry.30Hoover C.E. Davenport K.A. Henderson D.M. Pulscher L.A. Mathiason C.K. Zabel M.D. Hoover E.A. Detection and quantification of CWD prions in fixed paraffin embedded tissues by real-time quaking-induced conversion.Sci Rep. 2016; 6: 25098Crossref PubMed Scopus (20) Google Scholar In the current study, the next earliest time point was 30 dpi; thus, arrival of misfolded prion protein was between 30 and 60 dpi. Because the retina is spatially distinct from the brain, where the inoculum was delivered, we are confident that the seeding activity detected is a result of transport, presumably retrograde transport via the optic nerve31Walis A. Liberski P.P. Brown P. Ultrastructural alterations in the optic nerve in transmissible spongiform encephalopathies or prion diseases: a review.Folia Neuropathol. 2004; 42: 153-160PubMed Google Scholar; however, here we cannot entirely rule out transport by the circulatory system, as infectious prion protein was likely in blood of these animals before its detection in retina.32Sawyer E.B. Edgeworth J.A. Thomas C. Collinge J. Jackson G.S. Preclinical detection of infectivity and disease-specific PrP in blood throughout the incubation period of prion disease.Sci Rep. 2015; 5: 17742Crossref PubMed Scopus (14) Google Scholar Rapid retrograde transport of PrPSc in motor tracts, on the order of 6 weeks, has been previously reported after inoculation of the sciatic nerve.33Bartz J.C. Kincaid A.E. Bessen R.A. Retrograde transport of transmissible mink encephalopathy within descending motor tracts.J Virol. 2002; 76: 5759-5768Crossref PubMed Scopus (60) Google Scholar The consistent appearance of prion seeding activity in retinas at 60 dpi may suggest widespread distribution, albeit at a lower level than detected by traditional diagnostic methods, of misfolded prion protein at a relatively early stage of the disease process. The time course of some events (eg, PrPSc immunoreactivity) that we have reported in the retina is delayed by approximately 30 days compared to what has been reported in the brains of mice infected with RML scrapie.34Sandberg M.K. Al-Doujaily H. Sharps B. De Oliveira M.W. Schmidt C. Richard-Londt A. Lyall S. Linehan J.M. Brandner S. Wadsworth J.D. Clarke A.R. Collinge J. Prion neuropathology follows the accumulation of alternate prion protein isoforms after infective titre has peaked.Nat Commun. 2014; 5: 4347Crossref PubMed Scopus (91) Google Scholar We demonstrate detectable accumulation of PrPSc at 90 dpi. Interestingly, the earliest detectable accumulation of PrPSc was in the photoreceptor inner segments, rather than in retinal ganglion cells, whose axons comprise the optic nerve. Photoreceptors are at least two synaptic contacts removed from retinal ganglion cells, perhaps suggesting that accumulation of PrPSc there represents a tropism for misfolded protein accumulation in photoreceptors. The earliest detection of PrPSc accumulation was accompanied by a mild up-regulation of GFAP in Müller glia. This observation is consistent with our previous report of specific Müller glial activation in regions of early PrPSc accumulation in retinas of sheep with scrapie.13Greenlee J.J. Hamir A.N. West Greenlee M.H. Abnormal prion accumulation associated with retinal pathology in experimentally inoculated scrapie-affected sheep.Vet Pathology. 2006; 43: 733-739Crossref PubMed Scopus (31) Google Scholar Up-regulation of GFAP by Müller glia is the earliest indicator of retinal stress we have been able to detect and demonstrates a response by the retinal tissue to the presence of misfolded prion protein. Herein, we report widespread loss of photoreceptors in the retinas of mice with scrapie. This result is consistent with other rodent models of TSE, including a hamster model of transmissible mink encephalopathy35Buyukmihci N.C. Goehring-Harmon F. Marsh R.F. Photoreceptor degeneration during infection with various strains of the scrapie agent in hamsters.Exp Neurol. 1987; 97: 201-206Crossref PubMed Scopus (12) Google Scholar, 36Hogan R.N. Bowman K.A. Baringer J.R. Prusiner S.B. Replication of scrapie prions in hamster eyes precedes retinal degeneration.Ophthalmic Res. 1986; 18: 230-235Crossref PubMed Scopus (27) Google Scholar and mouse models of scrapie37Foster J. Farquhar C. Fraser J. Somerville R. Immunolocalization of the prion protein in scrapie affected rodent retinas.Neurosci Lett. 1999; 260: 1-4Crossref PubMed Scopus (16) Google Scholar or Creutzfeldt-Jakob disease.38Hogan R.N. Kingsbury D.T. Baringer J.R. Prusiner S.B. Retinal degeneration in experimental Creutzfeldt-Jakob disease.Lab Invest. 1983; 49: 708-715PubMed Google Scholar Our results demonstrate, however, that loss of these retinal neurons does not occur until approximately 80% of the incubation period, despite the fact that prion-seeding activity was detected in the retina at 40% of the incubation period. This observation is consistent with prior work demonstrating an increase in prion infectivity before the development of neuropathology.34Sandberg M.K. Al-Doujaily H. Sharps B. De Oliveira M.W. Schmidt C. Richard-Londt A. Lyall S. Linehan J.M. Brandner S. Wadsworth J.D. Clarke A.R. Collinge J. Prion neuropathology follows the accumulation of alternate prion protein isoforms after infective titre has peaked.Nat Commun. 2014; 5: 4347Crossref PubMed Scopus (91) Google Scholar Our results demonstrate the temporal separation of several key events in the pathogenesis of prion disease beginning with the transport of misfolded prion protein from the brain to the retina (Figure 6). The sequence of events described herein is not unexpected. However, by determining the time lag between these events in vivo (eg, the lag between PrPSc accumulation and cell death), we have provided concrete criteria that can be used not only to assess the effectiveness of anti-prion compounds,39Ghaemmaghami S. Russo M. Renslo A.R. Successes and challenges in phenotype-based lead discovery for prion diseases.J Med Chem. 2014; 57: 6919-6929Crossref PubMed Scopus (18) Google Scholar but also to determine the stage of disease that a given compound would be most effective. 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We thank Kevin Hassall, David Panthen, and Trudy Tatum for providing technical support to this project.