Detection and Quantification of CWD Prions in Fixed Paraffin Embedded Tissues by Real-Time Quaking-Induced Conversion

Hoover, Clare; Davenport, Kristen A.; Henderson, Davin M.; Pulscher, Laura A.; Mathiason, Candace K.; Zabel, Mark D.; Hoover, Edward A.

doi:10.1038/srep25098

Cited by 34 publications

(42 citation statements)

References 30 publications

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“…30 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 nerve 31 ; 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.…”

Section: Prion Seeding Activity Is Detected In the Retina Relativelymentioning

confidence: 64%

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

Greenlee

Lind

Kokemuller

et al. 2016

The American Journal of Pathology

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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 (PrP Sc ) from the brain to the retina, the accumulation of PrP Sc 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 PrP Sc and the resulting response of retinal tissue. Loss of photoreceptors was used as a measure of neuronal death. PrP Sc seeding activity was first detected in all samples at 60 dpi. Accumulation of PrP Sc 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 (PrP Sc ). 1 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. 2 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 PrP Sc 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, 3e8 development of effective therapies would be facilitated by a well-described in vivo model of misfolded protein transport and accumulation, with objective measures of neural degeneration.The retina is part of the CNS and is affected by numerous protein misfolding diseases, including Alzheimer disease, 9 Parkinson disease, 10e12 and numerous TSEs, including scrapie in sheep, 13,14 chronic wasting disease in

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Section: Prion Seeding Activity Is Detected In the Retina Relativelymentioning

confidence: 64%

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

Greenlee

Lind

Kokemuller

et al. 2016

The American Journal of Pathology

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“…RT-QuIC provides a rapid methodology for analysis of tissues and fluids containing minute quantities of prions (85,86) and may have utility in quantitative and correlative investigations of early and carrier states of prion diseases and potentially other protein misfolding disorders (86)(87)(88)(89). There is little to no evidence that RT-QuIC generates, or represents, the presence of prion infectivity.…”

Section: Discussionmentioning

confidence: 99%

“…Our previous studies have compared RT-QuIC seeding activity to CWD cervid brain in a 50% lethal dose (LD 50 ) bioassay (88,94). Because we used the same conditions and controls for this study, we applied this principle to our RT-QuIC data.…”

Section: Discussionmentioning

confidence: 99%

Infectious Prions in the Pregnancy Microenvironment of Chronic Wasting Disease-Infected Reeves' Muntjac Deer

Nalls

McNulty

Hoover

et al. 2017

J Virol

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Ample evidence exists for the presence of infectious agents at the maternal-fetal interface, often with grave outcomes to the developing fetus (i.e., Zika virus, brucella, cytomegalovirus, and toxoplasma). While less studied, pregnancyrelated transmissible spongiform encephalopathies (TSEs) have been implicated in several species, including humans. Our previous work has shown that prions can be transferred from mother to offspring, resulting in the development of clinical TSE disease in offspring born to muntjac dams infected with chronic wasting disease (CWD) (1). We further demonstrated protein misfolding cyclic amplification (PMCA)-competent prions within the female reproductive tract and in fetal tissues harvested from CWD experimentally and naturally exposed cervids (1, 2). To assess whether the PMCA-competent prions residing at the maternal-fetal interface were infectious and to determine if the real-time quaking-induced conversion (RT-QuIC) methodology may enhance our ability to detect amyloid fibrils within the pregnancy microenvironment, we employed a mouse bioassay and RT-QuIC. In this study, we have demonstrated RT-QuIC seeding activity in uterus, placentome, ovary, and amniotic fluid but not in allantoic fluids harvested from CWD-infected Reeves' muntjac dams showing clinical signs of infection (clinically CWD-infected) and in some placentomes from pre-clinically CWD-infected dams. Prion infectivity was confirmed within the uterus, amniotic fluid, and the placentome, the semipermeable interface that sustains the developing fetus, of CWD-infected dams. This is the first report of prion infectivity within the cervid pregnancy microenvironment, revealing a source of fetal CWD exposure prior to the birthing process, maternal grooming, or encounters with contaminated environments.IMPORTANCE The facile dissemination of chronic wasting disease within captive and free-range cervid populations has led to questions regarding the transmission dynamics of this disease. Direct contact with infected animals and indirect contact with infectious prions in bodily fluids and contaminated environments are suspected to explain the majority of this transmission. A third mode of transmission, from mother to offspring, may be underappreciated. The presence of pregnancyrelated prion infectivity within the uterus, amniotic fluid, and the placental structure reveals that the developing fetus is exposed to a source of prions long before exposure to the infectious agent during and after the birthing process or via contact with contaminated environments. These findings have impact on our current concept of CWD disease transmission.KEYWORDS CWD, maternal, pregnancy, prions T ransmissible spongiform encephalopathies (TSEs), or prion diseases, are proteinaceous infectious neurodegenerative diseases affecting animals, including humans (3, 4). The disease is caused by the accumulation of an aberrant misfolded form

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“…Our experiments performed during development of the FPE RT-QuIC method demonstrated that RT-QuIC amyloid seeding was achieved at higher seed concentrations of fixed paraffin-embedded (FPE) samples than had been previously reported using conventional frozen brain to prepare homogenates (15). Amyloid seeding in FPE brain-derived samples had a linear detection range extending from 10 Ϫ1 to 10 Ϫ7 , whereas non-FPE frozen brain homogenate samples displayed a linear range extending from 10 Ϫ4 to 10 Ϫ8 , likely due to inhibitors at high concentrations and consistent with published literature (Fig.…”

Section: Resultsmentioning

confidence: 93%

“…Through the development of methods to detect prion-seeding activity in fixed paraffin-embedded tissues using RT-QuIC (FPE RT-QuIC), we were able to detect amyloid-seeding activity at higher sample concentrations, 10 Ϫ1 and 10 Ϫ2 dilutions, than previously possible for brain homogenates (15). We then endeavored to identify the components of brain homogenate responsible for inhibiting the RT-QuIC amyloid formation reaction.…”

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confidence: 99%

Endogenous Brain Lipids Inhibit Prion Amyloid Formation In Vitro

Hoover

Davenport

Henderson

et al. 2017

J Virol

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The normal cellular prion protein (PrP C ) resides in detergent-resistant outer membrane lipid rafts in which conversion to the pathogenic misfolded form is believed to occur. Once misfolding occurs, the pathogenic isoform polymerizes into highly stable amyloid fibrils. In vitro assays have demonstrated an intimate association between prion conversion and lipids, specifically phosphatidylethanolamine, which is a critical cofactor in the formation of synthetic infectious prions. In the current work, we demonstrate an alternative inhibitory function of lipids in the prion conversion process as assessed in vitro by real-time quaking-induced conversion (RTQuIC). Using an alcohol-based extraction technique, we removed the lipid content from chronic wasting disease (CWD)-infected white-tailed deer brain homogenates and found that lipid extraction enabled RT-QuIC detection of CWD prions in a 2-log 10 -greater concentration of brain sample. Conversely, addition of brain-derived lipid extracts to CWD prion brain or lymph node samples inhibited amyloid formation in a dose-dependent manner. Subsequent lipid analysis demonstrated that this inhibitory function was restricted to the polar lipid fraction in brain. We further investigated three phospholipids commonly found in lipid membranes, phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol, and found all three similarly inhibited RT-QuIC. These results demonstrating polar-lipid, and specifically phospholipid, inhibition of prion-seeded amyloid formation highlight the diverse roles lipid constituents may play in the prion conversion process. IMPORTANCE Prion conversion is likely influenced by lipid interactions, given the location of normal prion protein (PrP C ) in lipid rafts and lipid cofactors generating infectious prions in in vitro models. Here, we use real-time quaking-induced conversion (RT-QuIC) to demonstrate that endogenous brain polar lipids can inhibit prionseeded amyloid formation, suggesting that prion conversion is guided by an environment of proconversion and anticonversion lipids. These experiments also highlight the applicability of RT-QuIC to identify potential therapeutic inhibitors of prion conversion.

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Detection and Quantification of CWD Prions in Fixed Paraffin Embedded Tissues by Real-Time Quaking-Induced Conversion

Cited by 34 publications

References 30 publications

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

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

Infectious Prions in the Pregnancy Microenvironment of Chronic Wasting Disease-Infected Reeves' Muntjac Deer

Endogenous Brain Lipids Inhibit Prion Amyloid Formation In Vitro

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