Oral Abstracts

Oral.01: Infectious full-length RecPrP-Fibrils: Insights from AFM-imaging and solid-state NMR-spectroscopy

Henrik Müller, Oleksandr Brener, Timo Piechatzek, Henrike Heise

Prions are known to be formed by β-sheet-rich amyloid fibrils. Their detailed structural architecture, however, is still obscure due to the failure of biophysical structure analysis on insoluble, non-crystalline and heterogeneous protein fibrils, not amenable to X-ray crystallography and liquid-state NMR. Since all model concepts available to date are based on low resolution data, it is a current matter of intense debate which part of the PrP-sequence forms the β-sheet core, whether prions contain β-helices¹ or β-sandwiches² and how the presence of infectious as well as non-infectious PrP-fibrils can be explained structurally. The rapid methodological developments of solid-state NMR in the last years established the possibility of applying NMR-spectroscopy also to solid biological macromolecules such as amyloid fibrils.³

In our contribution, we will report on our experimental progress in using high resolution solid-state NMR to structurally characterize amyloid fibrils of full-length ovine (ov) recombinant (rec) PrP(25–233). OvrecPrP-fibrils were formed spontaneously, by repeated seeding with pre-formed recPrP-fibrils as well as by seeding with Scrapie sheep brain-derived PrP^Sc in NMR-sufficient yields and demonstrated by biophysical characterization to closely resemble prions in living organisms. Bioassays in transgenic mice confirmed the infectious character of our ovrecPrP-fibrils.

All fibril preparations are characterized by enough homogeneity to draw first structural conclusions. First of all, our data are in agreement with a model for ovrecPrP-fibrils comprising a semi-mobile N-terminus, a middle part in the region between H114 and R154 comprising residues with chemical shifts indicative of α-helices or β-turns, and a distinct β-sheet core C-terminal of E155. The data are thus in line with the β-sandwich model. Second, although secondary structure elements are more or less identically arranged, PrP^Sc-seeding during fibrillation increases the conformational dynamics in some segments of the middle region providing an indication in which segment non-infectious recPrP-fibrils might undergo adaption in vivo to become infectious. Third, structural order in ovrecPrP-fibrils can be improved by selectively amplifying a more homogeneous fibril structure from a heterogeneous initial state using a repeated seeding protocol.

Oral.02: Coexistence and evolution of prions by natural selection

Chae Kim, Tracy Haldiman, Jan Langeveld, Qingzhong Kong, Jiri G. Safar

The unique phenotypic characteristics of mammalian prions are thought to be encoded in the conformation of pathogenic prion protein (PrP^Sc). Accordingly, a conformational mechanism is likely responsible for the evolution of prions observed in cloned cells. We hypothesized that the co-existence of similar conformers of PrP^Sc in the same prion isolate (strain) may be responsible for this cell-adaptation effect. Using biophysical techniques and conformation-dependent immunoassays in tandem, we isolated distinct subsets of PrP^Sc conformers with different conformational stabilities and aggregate sizes, which coexist in the most common human prion disease, sporadic Creutzfeldt-Jakob disease (sCJD). We did not observe an interaction between different conformers or the formation of hybrid aggregates in vitro. The serial protein misfolding cyclic amplification (PMCA) preferentially propagated only some of the conformers in the mixture, and led to the selection of those that had a lower conformational stability. Cumulatively, the data show that distinct subsets of independently replicating PrP^Sc conformers are present in sCJD and argue for a frequent coexistence of different prions. Under favorable conditions, the mixture of prions undergo evolution by natural selection of subset with the highest replication rate due to the lowest stability. These observations imply that:¹ most, if not all, of the natural (wild) prions and their strains are a dynamic collection of conformational subsets;² the fact that their stability shifts under new selection conditions may explain recently observed acquisition of drug resistance by mammalian and yeast prions.

Oral.03: De novo formation of recombinant prions by sPMCA

Zhihong Zhang, Fei Wang, Yi Zhang, Xinhe Wang, Guohua Yu, Yuanyuan Xu, Huaiyi Yang, Chonggang Yuan, Jiyan Ma

We previously showed that highly infectious prion can be generated with bacterially-expressed recombinant prion protein (PrP) by serial protein misfolding cyclic amplification (sPMCA), providing supporting evidence for the prion hypothesis. However, due to the powerful amplification capability of sPMCA, the origin of the recombinant prion, i.e., whether it was formed de novo or due to the amplification of inadvertently contaminated native prions, remains unclear. To address this question, we repeated the de novo recombinant prion formation in a newly established laboratory that has never been exposed to any native prions. Two proteinase K (PK) resistant, self-perpetuating recombinant PrP conformers (rPrP-res) were generated and the sizes of the PK-resistant cores were 17 kDa and 14 kDa. Both rPrP-res(17kDa) and rPrP^res(14kDa) were aggregated, contained a C-terminal PK-resistant core, and could be propagated indefinitely by sPMCA. Mouse bioassay revealed that the rPrP-res(17kDa) contained high infectivity, causing prion disease in wild-type mice with a 100% attack rate and a survival time around 172 d. In contrast, rPrP-res(14kDa) failed to cause disease in wildtype mice. Analyses of mice inoculated with infectious rPrP-res(17kDa) and rPrP-res(OSU) (recombinant prion previously generated via sPMCA at Ohio State University) revealed clear differences in the neuropathology caused by the two infectious recombinant prions. Biochemical analyses also showed that the two infectious recombinant prions differed in the size of PK-resistant fragment, the susceptibility to PK digestion, and the resistance to guanidine hydrochloride treatment. These results suggest that various PK-resistant self-propagating recombinant PrP conformers can be generated by sPMCA, but not all of them are capable of causing prion disease in wild-type animal. Moreover, generating an infectious prion in a prion-free environment supports that a recombinant prion can be formed de novo by sPMCA.

Oral.04: Infectious prions are associated with intact, morphologically distinct exosome vesicles

Andrew F. Hill, Belinda Guo, Lesley Cheng, Camelia Quek, Eric Hanssen, Victoria A. Lawson, Shayne A. Bellingham, Bradley M. Coleman

Prion diseases are associated with the misfolding of the cellular prion protein (PrP^C) into a conformation (PrP^Sc) that has transmissible properties. Recent studies have shown small cell derived nanovesicles called exosomes transfer prion infection between cells. We have used cryoelectron microscopy, infectivity bioassays, next generation sequencing from preparations of exosomes derived from control, prion infected, and cured infected cells to investigate the components of these vesicles required for the transmission of prion infection to uninfected cells. RNAi and chemical inhibitor approaches to knock down components of the exosome biogenesis pathways were used to determine their effects on prion infection. Cryo-EM tomography revealed that prion infection alters the ultrastructure of exosomes which is restored when the cells are cured, suggesting the infectious pathway affects the biogenesis of exosomes. Additionally, the miRNA profile of exosomes derived from prion infected cells is distinct to that of those from uninfected controls, suggesting that exosomes have potential as sources of biomarkers for prion infection. Furthermore, disruption of the structure of exosomes using chemical or physical methods affects the levels of prion infectivity, indicating intact vesicles are required for the successful transfer of infection. Together, these data also have implications for other neurodegenerative diseases such as Alzheimer and Parkinson disease in which the pathological proteins (Aβ and α-synuclein respectively) have also been associated with exosomes.

Oral.05: Contaminated blood products induce a highly atypical prion disease devoid of PrP^res in primates

Emmanuel Comoy, Nina Jaffré, Jacqueline Mikol, Valérie Durand, Christelle Jas-Duval, Sophie Luccantoni-Freire, Evelyne Correia, Nathalie Lescoutra-Etchegaray, Nathalie Streichenberger, Stéphane Haïk, Chryslain Sumian, Paul Brown, Jean-Philippe Deslys

Background

Concerns about the blood-borne risk of prion infection have been confirmed by the occurrence in the UK of four transfusion-related infections of vCJD and an apparently silent infection in an hemophiliac patient. Asymptomatic incubation periods in prion diseases can extend over decades in humans. We present here unexpected results of experiments evaluating blood transmission risk in a non-human primate model.

Material and Methods

Cynomolgus macaques were inoculated with brain or blood specimens from vCJD infected humans or monkeys. Neuropathological and biochemical findings were obtained using current methods used for human patients.

Results

Thirteen out of 23 primates exposed to various human or macaque blood products exhibited a previously undescribed myelopathic syndrome, devoid of the classical features of prion disease, notably abnormal prion protein (PrP^res) deposition, whereas the 14 corresponding brain-inoculated donor animals and 1 transfused animal exhibited the classical vCJD pattern. In passage experiments, plasma transfusion induced the same atypical phenotype after two years (again, with no detectable PrP^res), whereas the intracerebral inoculation of spinal cord led to a typical prion disease with cerebral spongiosis and PrP^res accumulation in the brain of the primate recipient. Interestingly, passage experiments in transgenic mice were largely unsuccessful.

In another experiment designed to test the efficacy of anti-prion filters, three recipients of filtered red blood cells suspended in plasma are still healthy 4.5 y after transfusion whereas the recipients of unfiltered inocula died after 2.5 y with the atypical neurological profile.

Conclusion

We describe a new fatal neurological myelopathic syndrome in monkeys exposed to various vCJD/BSE-infected blood components. Secondary transmission in primates confirms¹ the transmissibility of this myelopathy, and² its prion origin which could not be diagnosed as such in the first recipients. This myelopathy might be compared in some respects to certain forms of human lower motor neuron disease, including neuromyelitis optica, the flail arm syndrome of amyotrophic lateral sclerosis (ALS), and the recently described FOSMN (facial onset sensory and motor neuronopathy) syndrome.

Oral.06: Moose chronic wasting disease is efficiently transmitted to animals expressing deer and elk prion protein

Jeffrey R. Christiansen, Eri Saijo, Sehun Kim, Terry J. Kreeger, Glenn C. Telling

Background

Chronic wasting disease (CWD) is the only known transmissible spongiform encephalopathy (TSE) naturally affecting wildlife. Free-ranging deer and elk have been diagnosed with CWD throughout the Rocky Mountain region. Only recently were CWD infected moose identified, three in Colorado and one in Wyoming.¹ In addition, captive moose have been infected by oral inoculation of CWD infected mule deer brains.² Transmission of disease between cervid species has been predicted due to prion protein sequence homology, but experimental evidence of transmission from moose to deer or elk has been lacking.³ Understanding the transmission characteristics of CWD between all of the susceptible species sharing a habitat is crucial for the management of disease.

Material and Methods

Brain tissue from a CWD infected moose (Alces alces shirasi) was homogenized 1% w/v in PBS by passing through 18, 22 and 26 g syringes. Brain homogenates were intracranially inoculated into transgenic (Tg) mice expressing either deer or elk prion protein. Disease progression was monitored and animals were humanely euthanized at end-stage of disease. Collected brains were processed for immunoblotting, immunohistochemistry and histoblot evaluation.

Results

Transmission of CWD from moose to deer and elk was experimentally tested by Tg mouse bioassay. All of the inoculated animals showed clinical signs consistent with CWD infection. Disease incubation times of 322 ± 10 d were observed for Tg deer mice and 286 ± 16 d for Tg elk mice. Western blotting confirmed the presence of proteinase K (PK) resistant material from both study groups with glycoform ratios and migration patterns similar to deer derived CWD. Deposition of PrPsc was observed in brain regions consistent with previous observations of animals infected with type II CWD.⁴

Conclusion

Surveillance of harvested moose should continue in CWD endemic areas, as we did not observe the presence of a species barrier between members of the cervidae family. Behavioral differences, such as small social grouping, likely account for the low incidence rates of CWD in moose compared with infection rates observed in mule deer.

Oral.07: Rapid and sensitive antemortem detection of CWD prions in saliva of deer by RT-QuIC

Davin M. Henderson, Matteo Manca, Nicholas J. Haley, Nathaniel D. Denkers, Amy V. Nalls, Candace K. Mathiason, Byron Caughey, Edward A. Hoover

The hallmark of horizontally transmissible prion diseases may be the shedding/presence of infectious prions in saliva. Chronic wasting disease (CWD) is a prototype efficiently transmitted prion disease now identified in 22 United States, 2 Canadian provinces and Korea. Bioassays have demonstrated that infectious CWD prions are present in excreta, including especially saliva, but also urine and feces at lower concentrations. Nevertheless, in vitro detection of prions in these excreta [by, e.g., serial protein misfolding cyclic amplification (sPMCA)] has been problematic, apparently relating to inhibitors in the complex biofluids and excreta. Here we demonstrate rapid and sensitive detection of CWD prions in the saliva of pre-symptomatic and symptomatic CWDexposed whitetail deer by real-time quaking induced conversion (RTQuIC) in both unprocessed samples and using prion concentration enhancement. CWD prions were detected in untreated saliva from 6 of 11 pre-symptomatic and 5 of 9 clinical CWDexposed deer. Augmentation of the RTQuIC methodology by pre-seeding prion enrichment enhanced sensitivity, enabling prion detection in 16 of the 20 (80%) saliva samples from CWD-exposed whitetailed deer. In addition, Tg(cerPrP) mouse bioassay of 2 prototype RT-QuIC-positive saliva samples confirmed the presence of infectious prions in each. RTQuIC analysis promises to be a valuable means for non-invasive ante-mortem detection of CWD via biologic fluids, excreta, and tissues, and as well as for exploring the process of peripheralization and shedding of prions during CWD and other prion diseases.

Oral.08: Mother to offspring transmission of chronic wasting disease in Reeve’s Muntjac deer

Amy Nalls, Erin McNulty, Jenny Powers, Davis Seelig, Clare Hoover, Nicholas Haley, Jeanette Hayes-Klug, Kelly Anderson, Paula Stewart, Wilfred Goldmann, Edward A. Hoover, Candace K. Mathiason

To investigate the role mother to offspring transmission plays in chronic wasting disease (CWD), we have developed a cervid model employing the Reeve’s muntjac deer (Muntiacus reevesi). Eight muntjac doe were orally inoculated with CWD and tested PrP^CWD lymphoid positive by 4 mo post infection. Fourteen fawns were born to these eight CWD-infected doe—3 were born viable, 6 were born non-viable and 5 were harvested as fetuses from early or end-stage CWD-infected doe. All three viable fawns have demonstrated CWD IHC lymphoid biopsy positivity between 43 d post birth and 11 mo post birth. Two of these three CWD positive viable offspring have developed clinical signs consistent with TSE disease (28–33 mo post birth). Moreover, CWD prions have been detected by sPMCA in 11 of 16 tissues harvested from 6 full-term non-viable fawns and in 7 of 11 fetal tissues harvested in utero from the second and third trimester fetuses. Additional tissues and pregnancy related fluids from doe and offspring are being analyzed for CWD prions. In summary, using the muntjac deer model we have demonstrated CWD clinical disease in offspring born to CWD-infected doe, and in utero transmission of CWD from mother to offspring. These studies provide basis to further investigate the mechanisms of maternal transfer of prions.

Oral.09: Efficient transmission of diverse prion strains to transgenic mice expressing bank vole prion protein

Joel C. Watts, Kurt Giles, Smita Patel, Abby Oehler, Stephen J. DeArmond, Stanley B. Prusiner

The interspecies transmission of prions is typically an inefficient process that is characterized by low transmission rates and long incubation periods, a phenomenon referred to as the species barrier. Interestingly, despite expressing a prion protein (PrP) sequence that differs from mouse PrP at only eight positions, bank voles exhibit an unprecedented susceptibility to a diverse range of prion strains isolated from multiple different species. To determine if this enhanced susceptibility to prions is encoded within the sequence of bank vole PrP (BVPrP), we inoculated transgenic (Tg) mice expressing BVPrP containing either the methionine or isoleucine polymorphism at codon 109 with a diverse range of prion strains. Tg(BVPrP,M109) mice were susceptible to the following prion isolates: mouse-adapted scrapie strain RML, mouse-adapted bovine spongiform encephalopathy (BSE) strain 301V, hamster-adapted scrapie strain Sc237, sheep scrapie isolate SSBP/1, elk chronic wasting disease (CWD), cattle BSE, sporadic Creutzfeldt-Jakob disease (CJD) subtypes MM1, MM2, and VV2, as well as variant CJD. All transmissions resulted in neuropathological changes indicative of prion disease, including spongiform change, PrP deposition, and the accumulation of proteinase K (PK)-resistant PrP. The strain properties of the various prion isolates were generally maintained upon serial passage in Tg(BVPrP,M109) mice, as judged by the electrophoretic mobility of PK-resistant PrP, patterns of cerebral PrP deposition and guanidine stability measurements. Furthermore, retrotransmission of BVPrP-adapted RML, Sc237, and sporadic CJD MM1 prions to Tg mice expressing mouse, hamster, or human PrP, respectively, resulted in re-isolation of the original strain. Tg(BVPrP,I109) mice were also susceptible to a diverse set of prion strains. However, transmission of elk CWD or sporadic CJD MM1 prions to Tg(BVPrP,I109) mice resulted in incubation periods that partially overlapped the onset of spontaneous disease in these mice. Multiple distinct prion strains were observed in CWD- and CJD-inoculated Tg(BVPrP,I109) mice, suggesting that the presence of spontaneously formed misfolded PrP conformers can alter the fidelity of prion strain replication. Our results demonstrate that expression of BVPrP is sufficient to engender enhanced susceptibility to a diverse range of prion strains and that, at most, eight residues in PrP mediate this phenomenon.

Oral.10: Synthetic human α-synuclein prions

Tran Thanh Nhat Le, Hoang Ngoc Ai Tran, Suzana Aulic, Fabio Moda, Paolo Zucca, Fabrizio Tagliavini, Stefano Gustincich, Giuseppe Legname

Numerous findings from cell cultures and transgenic mice experiments are suggestive of intercellular α−synuclein transfer between cells. This mechanism may contribute to the spreading of the neuropathology in Parkinson Disease (PD). In fact, in PD, β-sheet-rich α-synuclein may cross from the transplanted patient’s own neurons into grafted cells and induce α-synuclein to acquire a β-sheet-rich structure.

We applied a methodology used to obtain synthetic prions in order to test whether synthetic pathological agents such as recombinant human α-synuclein amyloids are able to infect neuronal cell lines in vitro and wild-type mice in vivo.

A single exposure to amyloid fibrils of recombinant human α-synuclein was sufficient to induce aggregation of endogenous α-synuclein in human neuroblastoma SH-SY5Y cells. Interestingly, we found pathological α-synuclein (phosphorylated at Serine 129) in amyloid-infected cells.

Our results suggest that recombinant α-synuclein amyloids can promote endogenous α-synuclein aggregation and pathological post-translational modification. Mice inoculated in the substantia nigra pars compacta with recombinant human α-synuclein amyloid showed marked neurological symptoms resembling those of PD, upon several passages.

Oral.11: Variant CJD 17 years on

Jean C. Manson, Matthew Bishop, Abigail B. Diack, Diane Ritchie, Sandra McCutcheon, Richard Alejo Blanco, Pedro Piccardo, James Ironside, Robert Will

It is now 17 years since the first identification of a case of vCJD in the UK. Since that time there has been much speculation over how vCJD might impact on human health. To date there have been 176 cases reports in the UK and a further 51 cases worldwide in 11 different countries. It has been important to establish (1) if all worldwide cases represent the same strain of agent; (2) the potential for human to human transmission; (3) if the strain of agent will adapt to the human host and become more virulent; and (4) the extent to which asymptomatic infection may impact on human health.

We have now established by examining a number of worldwide vCJD cases that there is broad similarity to UK vCJD cases on first passage to mice. Transgenic mouse studies have indicated that all codon 129 genotypes are susceptible to vCJD and that genotype may influence whether disease appears in a clinical or asymptomatic form, supported by the appearance of the first case of asymptomatic vCJD infection in a PRNP 129MV patient. We have established, by transmission to RIII mice, that there is little evidence for strain modification following human to human transmission of vCJD by blood transfusion of an MM case to an MM or MV recipient. Some differences in incubation times in VM mice observed in these transmissions are now being assessed on second passage to establish if alterations represent differences in strain characteristics or are due to species barrier effects in primary passage. Following evidence of blood transfusion as a route of transmission, we have ascertained that all blood components and leucoreduced blood, in a sheep model of vCJD have the ability to transmit disease. Importantly, we have recently established that a blood recipient with an asymptomatic infection with limited PrP^Sc deposition in the spleen could readily transmit disease into mice, demonstrating the potential for peripheral infection in the absence of clinical disease. This, along with the recent appendix survey which identified 16 positive appendices in a study of 32,441 cases, underlines the importance of continued CJD surveillance and maintaining control measures already in place to protect human health.

Oral.12: Preclinical detection of variant CJD and BSE prions in blood

Caroline Lacroux, Jean Yves Douet, Emmanuel Comoy, Hugh Simmons, Vincent Beringue, Jean Philippe Deslys, Didier Vilette, Olivier Andreoletti

The emergence of variant Creutzfeldt Jakob Disease (vCJD) is considered a likely consequence of human dietary exposure to Bovine Spongiform Encephalopathy (BSE) agent. More recently, secondary vCJD cases were identified in patients transfused with blood products prepared from apparently healthy donors who later went on to develop the disease. As there is no validated assay for detection of vCJD/BSE infected individuals the prevalence of the disease in the population remains uncertain. In that context the risk of vCJD blood borne transmission is considered as a serious concern by health authorities.

In this study, appropriate conditions and substrates for highly efficient and specific in vitro amplification of vCJD/BSE agent using Protein Misfolding Cyclic Assay (PMCA) were first identified. This showed that whatever the origin (species) of vCJD/BSE agent, the ovine Q171 PrP substrates provided the best amplification performances. These results indicate that the homology of PrP amino-acid sequence between the seed and the substrate is not the crucial determinant of the vCJD agent in vitro propagation.

The ability of this method to detect endogenous vCJD/BSE agent in the blood was then defined. In both sheep and primate models of the disease, the assay enabled the identification of infected individuals in the early preclinical stage of the incubation period. Finally, blood from two vCJD affected patients and 135 healthy controls were tested. The assay detected the presence of the vCJD case within a pool of several dozens of human blood samples. The equivalent 0.05 µL of whole blood from the vCJD affected patient was sufficient for amplifying PrP^res.These results open new possibilities for vCJD screening and prevention of its iatrogenic transmission.

Oral.13: Prion detection in urine of patients with variant Creutzfeldt-Jakob disease

Fabio Moda, Silvio Notari, Pierluigi Gambetti, Valeria Fugnanesi, Kyung-Won Park, Michela Morbin, Silvia Suardi, Fabrizio Tagliavini, Claudio Soto

Definitive diagnosis of prion disease can only be made by tissue examination and relies on the detection of misfolded prion protein (PrP^Sc) and associated histopathological changes in the brain. PrP^Sc is the main component of the infectious agent and is the only validated surrogate marker for the disease. The unique mechanism of prion transmission and the appearance of a new variant form of CJD (vCJD), which has been linked to the consumption of meat from BSE-affected cattle, have raised concerns for public health. Recently, four cases of vCJD have been associated with blood transfusion from asymptomatic donors who subsequently died from vCJD. This suggests that prions exist in the blood of individuals silently incubating the disease. It is likely that minute amount of prions can be present in urine of vCJD patients, as product of blood filtration, but no evidences of prion detection in urine of humans with CJD have been provided so far. With the advent of PMCA (protein mysfolding cyclic amplification) several groups have been able to detect infectious prion in urine of cervids infected by chronic wasting disease (CWD), sheep infected by Scrapie and rodents (mouse and hamsters) infected with different prion agents. Taking advantage of the extreme sensitivity of PMCA, we have analyzed urine from several patients suffering from different forms of CJD (variant, sporadic and genetic). Of all the cases analyzed, only those affected by vCJD were found to contain PrP^Sc that can be amplified to obtain a signal that has the typical electrophoretic profile of PrP^Sc associated to vCJD. Many controls including urine from patients affected by other neurological diseases (Alzheimer disease, Frontotemporal dementia, Parkinson disease, progressive supranuclear palsy), as well as healthy control subjects were all found to be negative for PrP^Sc in urine. To the best of our knowledge, this is the first report showing the presence of prions in human urine. Due to the limited number of vCJD urine samples examined, we cannot definitively establish whether PrP^Sc presence is common in all vCJD patients or depends on the existence of other pathologies (e.g., renal malfunction). The detection of prions in human urine may be utilized as a non-invasive diagnostic test of vCJD, and also uncovers a possible risk related with the use of urinary-derived products (hormones, enzymes and other proteins) as well as the collection and disposal of urine from vCJD patients.

Acknowledgments

This research was partially fund by NIH grants R42NS079060, P01AI077774 and P01AG14359 to CS and P01AG14359 and Charles S. Britton Fund to PG.

Oral.14: A systems approach to frontotemporal dementia

Inyoul Lee, Taek-Kyun Kim, Ji-Hoon Cho, David Baxter, Kelsey Scherler, Brenda Canine, Rose Pitstick, Daehee Hwang, George Carlson, Leroy Hood

Frontotemporal dementia (FTD) is characterized by neurofibrillary tangles (NFTs) composed of misfolded and aggregated tau protein that are associated with neuronal death and dementia. Despite the availability of well-studied mouse models for FTD, the molecular mechanisms underlying FTD still needs to be dissected. Here, we present a systems approach that enables us to: (1) capture significant molecular signatures associated with tauopathy and (2) construct human mutant tau perturbed networks reflecting brain sub-region specificity. We compared two bigenic transgenic mouse lines: rTg4510 that overexpresses human P301L mutant tau transgene with rTg21221 that overexpresses human wild type tau, at four time points spanning from 30 d to 210 d. We applied an integrative statistical hypothesis test and identified 842 differentially expressed genes (DEGs): (1) early upregulated genes, (2) late upregulated genes, (3) early downregulated genes and (4) late downregulated genes. Using the 842 DEGs, gene ontology classification and known protein interactions, we constructed hypothetical molecular networks perturbed at different stages of tau hyperphosphorylation and NFT formation, in order to delineate key cellular processes and transcriptional regulations associated with tauopathy in FTD. The constructed network shows that dynamic changes of genes associated with pathological features as the mice age. Networks involving immune response and synaptic degeneration were revealed and genes involved in mitochondrial and oxidative stress also were identified. Gene expression profiles in 26 brain sub-regions from Allen Brain Atlas, indicated that the DEGs were highly expressed in cerebral cortex and hippocampus regions. An integration of the expression profiles revealed dynamic transition of gene expression and key biological processes. This systems approach provides novel insights in FTD pathogenesis.

Oral.15: Molecular barriers to zoonotic prion transmission: Comparison of the ability of sheep, cattle and deer prion disease isolates to convert normal human prion protein to its pathological isoform in a cell-free system

Marcelo A. Barria, Aru Balachandran, Masanori Morita, Tetsuyuki Kitamoto, Rona Barron, Jean Manson, Richard Knight, James W. Ironside, Mark W. Head

Background

Bovine spongiform encephalopathy (BSE) is a known zoonotic prion disease, resulting in variant Creutzfeldt-Jakob disease (vCJD) in humans. In contrast, classical scrapie in sheep is thought to offer little or no danger to human health. However, a widening range of prion diseases have been recognized in cattle, sheep and deer. The risks posed by individual animal prion diseases to human health cannot be determined a priori and are difficult to assess empirically. The fundamental event in prion disease pathogenesis is thought to be the seeded conversion of normal prion protein (PrP^C) to its pathological isoform (PrP^Sc). Here we report the use of a rapid molecular conversion assay to test whether brain specimens from different animal prion diseases are capable of seeding the conversion of human PrP^C to PrP^Sc.

Material and Methods

Classical BSE (C-type BSE), H-type BSE, L-type BSE, classical scrapie, atypical scrapie, chronic wasting disease and vCJD brain homogenates were tested for their ability to seed conversion of human PrP^C to PrP^Sc in protein misfolding cyclic amplification (PMCA) reactions. Newly formed human PrP^Sc was detected by protease digestion and western blotting using the antibody 3F4.

Results

C-type BSE and vCJD were found to efficiently convert PrP^C to PrP^Sc. Scrapie failed to convert human PrP^C to PrP^Sc. Of the other animal prion diseases tested only chronic wasting disease appeared to have the capability to convert human PrP^C to PrP^Sc. The results were consistent whether the human PrP^C came from human brain, humanised transgenic mouse brain or from cultured human cells and the effect was more pronounced for PrP^C with methionine at codon 129 compared with that with valine.

Conclusion

Our results show that none of the tested animal prion disease isolates are as efficient as C-type BSE and vCJD in converting human prion protein in this in vitro assay. However, they also show that there is no absolute barrier to conversion of human prion protein in the case of chronic wasting disease.

Oral.16: Is cancer a prion disease? Prion-like properties of amyloid oligomers and fibrils of mutant p53

Jerson L. Silva, Ana P. Ano Bom, Danielly C. Costa, Guilherme A. de Oliveira, Yraima Cordeiro, Claudia V. De Moura Gallo, Luciana P. Rangel

The tumor suppressor protein p53 loses its function in more than 50% of all human cancers. It has been postulated that mutant p53 can form aggregates that are related to loss-of-function and cancer development.^1-4 Also, new data have contributed to spread the application of the prion concept to different neurodegenerative and amyloid disesases.^5-7 To evaluate the role of aggregation in cancer, we investigated whether wild-type (WT) p53 and the hot-spot mutant R248Q could aggregate as amyloids under physiological conditions and whether the mutant could seed aggregation of the wild-type form.⁸ The central domains (p53C) of both constructs aggregated into a mixture of oligomers and fibrils. R248Q had a greater tendency to aggregate than WT p53. Full-length p53 aggregated into amyloid-like species that bound thioflavin T. The amyloid nature of the aggregates was evaluated using X-ray diffraction, electron microscopy, FTIR, dynamic light scattering, cell viabilility assay, congo red binding and its green birefringence under polarized light and anti-amyloid immunoassay. The X-ray diffraction pattern of the fibrillar aggregates was consistent with the typical conformation of cross β-sheet amyloid fibers with reflexions of 4.7 Å and 10 Å. A seed of R248Q p53C amyloid oligomers and fibrils resulted in acceleration of the aggregation of WT p53C, a behavior typical of a prion. As shown previously for other mutants,⁹ the R248Q mutant co-localized with amyloid-like species in a breast cancer sample, which further supported its prion-like effect. A tumor cell line containing mutant p53 also revealed massive aggregation of p53 in the nucleus. We conclude that aggregation of p53 into a mixture of oligomers and fibrils sequestrates the native protein into an inactive conformation that is typical of a prionoid. This prion-like behavior of oncogenic p53 mutants provides an explanation for the negative dominance effect observed in cancer. We are also studying the effects of different compounds targeting p53 mutant aggregation that are potential anticancer drugs.

Oral.17: Toxic misfolded prion protein causes neuronal death by NAD⁺ starvation

Minghai Zhou, Gregory Ottenberg, Gian Franco Sferrazza, Christopher Hubbs, Mohammad Fallahi, Gavin Rumbaugh, Alicia Brantley, Corinne I. Lasmézas

The mechanisms of neuronal death in protein misfolding neurodegenerative diseases (PMNDs) such as Alzheimer, Parkinson and prion diseases are poorly understood. We used our highly toxic misfolded prion protein (TPrP) model to understand prion protein neurotoxicity. We show that TPrP-induced apoptosis and autophagy are not the primary causes of neuronal demise. On the other hand, TPrP-damaged neuroblastoma cells and primary neurons were completely rescued by treatment with nicotinamide adenine dinucleotide (NAD⁺) or its precursor nicotinamide (NAM). TPrP-exposed cells were severely NAD⁺ depleted and NAD⁺ or NAM treatment restored intracellular NAD⁺ pool. Intracerebral injection of NAD⁺ dose-dependently protected hippocampal neurons from TPrP-induced degeneration. Intranasal NAD⁺ treatment of prion-infected mice starting during established clinical phase significantly delayed motor impairment and reduced the time during which mice were non-ambulatory. We identified for the first time NAD⁺ starvation as the cause of neurodegeneration induced by a misfolded amyloidogenic protein and as an autophagy inducer in a PMND. We propose the development of NAD⁺ replenishment strategies for neuroprotection in prion diseases and possibly other PMNDs.