Circulation of Nor98 Atypical Scrapie in Portuguese Sheep Confirmed by Transmission of Isolates into Transgenic Ovine ARQ-PrP Mice

Portugal was among the first European countries to report cases of Atypical Scrapie (ASc), the dominant form of Transmissible Spongiform Encephalopathy (TSE) in Portuguese small ruminants. Although the diagnostic phenotypes observed in Portuguese ASc cases seem identical to those described for Nor98, unequivocal identification requires TSE strain-typing using murine bioassays. In this regard, we initiated characterization of ASc isolates from sheep either homozygous for the ARQ genotype or the classical scrapie-resistant ARR genotype. Isolates from such genotypes were transmitted to TgshpXI mice expressing ovine PrPARQ. Mean incubation periods were 414 ± 58 and 483 ± 107 days in mice inoculated with AL141RQ/AF141RQ and AL141RR/AL141RR sheep isolates, respectively. Both isolates produced lesion profiles similar to French ASc Nor98 ‘discordant cases’, where vacuolation was observed in the hippocampus (G6), cerebral cortex at the thalamus (G8) level, cerebellar white matter (W1) and cerebral peduncles (W3). Immunohistochemical PrPSc deposition was observed in the hippocampus, cerebellar cortex, cerebellar white matter and cerebral peduncles in the form of aggregates and fine granules. These findings were consistent with previously reported cases of ASc Nor98 transmitted to transgenic TgshpXI mice, confirming that the ASc strain present in Portuguese sheep corresponds to ASc Nor98.

PMCA-generated prions from the olfactory mucosa of patients with Fatal Familial Insomnia cause prion disease in mice

Background:Fatal Familial Insomnia (FFI) is a genetic prion disease caused by the D178N mutation in the prion protein gene (PRNP) in coupling phase with methionine at PRNP 129. In 2017, we have shown that the olfactory mucosa (OM) collected from FFI patients contained traces of PrPSc detectable by Protein Misfolding Cyclic Amplification (PMCA).Methods:In this work, we have challenged PMCA-generated products obtained from OM and brain homogenate of FFI patients in BvPrP-Tg407 transgenic mice expressing the bank vole prion protein to test their ability to induce prion pathology.Results:All inoculated mice developed mild spongiform changes, astroglial activation, and PrPSc deposition mainly affecting the thalamus. However, their neuropathological alterations were different from those found in the brain of BvPrP-Tg407 mice injected with raw FFI brain homogenate.Conclusions:Although with some experimental constraints, we show that PrPSc present in OM of FFI patients is potentially infectious.Funding:This work was supported in part by the Italian Ministry of Health (GR-2013-02355724 and Ricerca Corrente), MJFF, ALZ, Alzheimer’s Research UK and the Weston Brain Institute (BAND2015), and Euronanomed III (SPEEDY) to FM; by the Spanish Ministerio de Economía y Competitividad (grant AGL2016-78054-R [AEI/FEDER, UE]) to JMT and JCE; AM-M was supported by a fellowship from the INIA (FPI-SGIT-2015-02).

Prion-induced photoreceptor degeneration begins with misfolded prion protein accumulation in cones at two distinct sites: cilia and ribbon synapses

Accumulation of misfolded host proteins is central to neuropathogenesis of numerous human brain diseases including prion and prion-like diseases. Neurons of retina are also affected by these diseases. Previously, our group and others found that prion-induced retinal damage to photoreceptor cells in mice and humans resembled pathology of human retinitis pigmentosa caused by mutations in retinal proteins. Here, using confocal, epifluorescent and electron microscopy we followed deposition of disease-associated prion protein (PrPSc) and its association with damage to critical retinal structures following intracerebral prion inoculation. The earliest time and place of retinal PrPSc deposition was 67 days post-inoculation (dpi) on the inner segment (IS) of cone photoreceptors. At 104 and 118 dpi, PrPSc was associated with the base of cilia and swollen cone inner segments, suggesting ciliopathy as a pathogenic mechanism. By 118 dpi, PrPSc was deposited in both rods and cones which showed rootlet damage in the IS, and photoreceptor cell death was indicated by thinning of the outer nuclear layer. In the outer plexiform layer (OPL) in uninfected mice, normal host PrP (PrPC) was mainly associated with cone bipolar cell processes, but in infected mice, at 118 dpi, PrPSc was detected on cone and rod bipolar cell dendrites extending into ribbon synapses. Loss of ribbon synapses in cone pedicles and rod spherules in the OPL was observed to precede destruction of most rods and cones over the next 2–3 weeks. However, bipolar cells and horizontal cells were less damaged, indicating high selectivity among neurons for injury by prions. PrPSc deposition in cone and rod inner segments and on the bipolar cell processes participating in ribbon synapses appear to be critical early events leading to damage and death of photoreceptors after prion infection. These mechanisms may also occur in human retinitis pigmentosa and prion-like diseases, such as AD.

Partial dissociation of PrPSc deposition and vacuolation in the brains of scrapie and BSE experimentally affected goats

The diagnosis of transmissible spongiform encephalopathies (TSEs) depends on the detection of vacuolation in brain sections taken from affected individuals and/or the identification of the disease-associated isoform of the PrP (prion) protein (PrPSc). During the course of an investigation, goats clinically affected following experimental infection with three different sources of TSE (SSBP/1, CH1641 and BSE) developed widespread vacuolar degeneration in the brain. With BSE, PrPSc was clearly recognized in affected goat brain by immunocytochemistry (icc) and Western blotting, but in contrast the experimental scrapie sources SSBP/1 and CH1641 showed almost no or very little PrPSc by icc. Western blot analysis of PrPSc from BSE-affected and SSBP/1-affected goat brain showed that the protein was present in brain affected by both TSE sources, but could not be used to determine how much protein was present. It became clear that PrPSc and vacuolation could be partially dissociated following challenge with two of the three TSE sources. Subtle differences in glycosylation patterns between BSE- and SSBP/1-associated PrP protein isoforms could also be recognized, although these experimentally generated results should not be regarded as a BSE/scrapie differential test. However, our study warns that the reliance on PrPSc determination by icc alone as a means by which to diagnose TSE infection may generate false negative results.