<figure><img src="image_136.png" /></figure>

School of Biotechnology, Jawaharlal Nehru University, New Delhi, USA

CONTACT Nikita Admane niki.admane@gmail.com

## ABSTRACT

Misfolding and aggregation of human prion protein (HuPrP) is a hallmark to a variety of neurological disorders, commonly known as the Transmissible Spongiform Encephalopathies (TSEs). Despite several efforts to recognize effective small molecule inhibitors of amyloid disorders, presently no effective therapeutic agent is available for the neurodengerative amyloidosis. Herein, we examined the protective efficacy of two green tea polyphenols, GTP1 and GTP2 against the amyloid conversion of cellular human prion protein to the disease forming scrapie conformation, employing a combination of computational, and experimental approach. Collective results from ThT, TEM, AFM, DLS, and CD experiments show that both the GTPs exhibit an inhibitory effect on the structural transition of HuPrP monomers into beta sheet rich structures, thereby reducing the amyloid fibrillization of HuPrP. Further, MTT assay and confocal microscopy revealed that both the catechins increase the cell viability by reducing toxicity associated with oligomeric species in neuronal cell models. But interestingly, GTP1 has a stronger inhibitory effect on HuPrP amyloidogenesis as compared to GTP2 due to minor differences in the structure of these two polyphenols. Both the compounds stably interact with the hydrophobic residues in the binding pocket of HuPrP which encompasses the β2-α2 loop and the C-terminal region of the α3 helix. Binding mechanisms of GTP1 and GTP2 with HuPrP was analysed by molecular dynamics simulations. The 4Å region of HuPrP binding pocket around these two compounds reveals that GTP1 forms more hydrogen bonds and hydrophobic interactions (as compared to GTP2) with the residues critical in the conversion of cellular to scrapie form of HuPrP, this may be the reason behind making GTP1 a more potent inhibitor. Thus, small molecules (like catechins and polyphenols) that can directly interact with the critical hydrophobic patches of HuPrP can act as inhibitors of prion amyloidogenesis and hold a substantial therapeutic potential. The green tea polyphenols might also be of synergistic benefit in treating the prion diseases.

## 244. The use of recreational hunters in Chronic Wasting Disease (CWD) management: behaviour and incentives

Lusi Xieª, Vic Adamowiczª and Patrick Lloyd-Smithᵇ

ªUniversity of Alberta; ᵇUniversity of Saskatchewan

CONTACT Lusi Xie lxie@ualberta.ca

## ABSTRACT

**Background:** While many jurisdictions in North America have implemented some management programmes to address Chronic Wasting Disease (CWD), the disease continues to spread. Although epidemiological models have proposed depopulating infected animals to reduce disease prevalence and spread by increasing hunter harvest (e.g. Potapov et al. 2016) [1], these models do not evaluate the incentives required to increase hunter harvest or the impacts of wildlife disease and management programmes on recreational activity and value. A better understanding of human behavioural responses will help effectively incorporate recreational hunters in wildlife disease control. The objective of this study is to examine the use of incentives to recreational hunters for CWD management by studying hunting trip decisions in response to CWD and its management. We develop an empirical model, with a focus on spatial and temporal substitution patterns of recreational activities, as well as economic benefits of a new recreation season.

**Materials and Methods:** The data used in this study are from an online survey of recreational hunters in Alberta and Canada. Five thousand hunters were sampled and a response rate of approximately 20% resulted in a usable sample of 707. Hunters were asked to recall hunting trips in 2017 and stated intended visitation with proposed extended hunting seasons for CWD management. We develop a discrete-continuous empirical model that captures hunting trip decisions on sites, periods and frequencies. Explanatory variables included in the model are CWD prevalence levels, policy dummy variables, individuals' socio-demographic variables as well as travel and time costs. With the estimated parameters, spatial and temporal substitution patterns are captured, measures of the potential harvest of affected deer populations, and economic benefits, are calculated.

**Results and Conclusions:** We find a negative but insignificant coefficient of CWD which indicates that individuals do not avoid hunting in CWD-infected areas. Individuals are likely to take around six additional trips on average during the extended seasons – this is more than half of the average trips (around 10) they actually took in 2017. We find that individuals are likely to substitute from hunting in areas with lower CWD risks to hunting in the most CWD-infected areas in both seasons. Individuals are better off from an extended season for CWD management. Our findings
<figure><img src="image_140.png" /></figure>

School of Biotechnology, Jawaharlal Nehru University, New Delhi, USA

CONTACT Nikita Admane niki.admane@gmail.com

## ABSTRACT

Misfolding and aggregation of human prion protein (HuPrP) is a hallmark to a variety of neurological disorders, commonly known as the Transmissible Spongiform Encephalopathies (TSEs). Despite several efforts to recognize effective small molecule inhibitors of amyloid disorders, presently no effective therapeutic agent is available for the neurodengerative amyloidosis. Herein, we examined the protective efficacy of two green tea polyphenols, GTP1 and GTP2 against the amyloid conversion of cellular human prion protein to the disease forming scrapie conformation, employing a combination of computational, and experimental approach. Collective results from ThT, TEM, AFM, DLS, and CD experiments show that both the GTPs exhibit an inhibitory effect on the structural transition of HuPrP monomers into beta sheet rich structures, thereby reducing the amyloid fibrillization of HuPrP. Further, MTT assay and confocal microscopy revealed that both the catechins increase the cell viability by reducing toxicity associated with oligomeric species in neuronal cell models. But interestingly, GTP1 has a stronger inhibitory effect on HuPrP amyloidogenesis as compared to GTP2 due to minor differences in the structure of these two polyphenols. Both the compounds stably interact with the hydrophobic residues in the binding pocket of HuPrP which encompasses the β2-α2 loop and the C-terminal region of the α3 helix. Binding mechanisms of GTP1 and GTP2 with HuPrP was analysed by molecular dynamics simulations. The 4Å region of HuPrP binding pocket around these two compounds reveals that GTP1 forms more hydrogen bonds and hydrophobic interactions (as compared to GTP2) with the residues critical in the conversion of cellular to scrapie form of HuPrP, this may be the reason behind making GTP1 a more potent inhibitor. Thus, small molecules (like catechins and polyphenols) that can directly interact with the critical hydrophobic patches of HuPrP can act as inhibitors of prion amyloidogenesis and hold a substantial therapeutic potential. The green tea polyphenols might also be of synergistic benefit in treating the prion diseases.

## 244. The use of recreational hunters in Chronic Wasting Disease (CWD) management: behaviour and incentives

Lusi Xieª, Vic Adamowiczª and Patrick Lloyd-Smithᵇ

ªUniversity of Alberta; ᵇUniversity of Saskatchewan

CONTACT Lusi Xie lxie@ualberta.ca

## ABSTRACT

**Background:** While many jurisdictions in North America have implemented some management programmes to address Chronic Wasting Disease (CWD), the disease continues to spread. Although epidemiological models have proposed depopulating infected animals to reduce disease prevalence and spread by increasing hunter harvest (e.g. Potapov et al. 2016) [1], these models do not evaluate the incentives required to increase hunter harvest or the impacts of wildlife disease and management programmes on recreational activity and value. A better understanding of human behavioural responses will help effectively incorporate recreational hunters in wildlife disease control. The objective of this study is to examine the use of incentives to recreational hunters for CWD management by studying hunting trip decisions in response to CWD and its management. We develop an empirical model, with a focus on spatial and temporal substitution patterns of recreational activities, as well as economic benefits of a new recreation season.

**Materials and Methods:** The data used in this study are from an online survey of recreational hunters in Alberta and Canada. Five thousand hunters were sampled and a response rate of approximately 20% resulted in a usable sample of 707. Hunters were asked to recall hunting trips in 2017 and stated intended visitation with proposed extended hunting seasons for CWD management. We develop a discrete-continuous empirical model that captures hunting trip decisions on sites, periods and frequencies. Explanatory variables included in the model are CWD prevalence levels, policy dummy variables, individuals' socio-demographic variables as well as travel and time costs. With the estimated parameters, spatial and temporal substitution patterns are captured, measures of the potential harvest of affected deer populations, and economic benefits, are calculated.

**Results and Conclusions:** We find a negative but insignificant coefficient of CWD which indicates that individuals do not avoid hunting in CWD-infected areas. Individuals are likely to take around six additional trips on average during the extended seasons – this is more than half of the average trips (around 10) they actually took in 2017. We find that individuals are likely to substitute from hunting in areas with lower CWD risks to hunting in the most CWD-infected areas in both seasons. Individuals are better off from an extended season for CWD management. Our findings
<figure><img src="image_137.png" /></figure>

indicates that it is possible to direct hunting trips for CWD control by extending hunting seasons in CWD-infected areas without additional monetary or non-monetary incentives.

**KEYWORDS:** Chronic wasting disease; recreation demand

## Reference

[1] Potapov, et al. PLoS One. 2016;11(3):e0151039.

## 245. PrPC knockdown by Liposome-siRNA-peptide complexes (LSPCs) prolongs survival and normal behaviour in prion-infected mice immunotolerant to treatment

Heather Bender<sup>a,b</sup>, Noelle Noyes<sup>a,c</sup>, Jessica L Annis<sup>a</sup>, Amanda Hitpas<sup>a</sup>, Luke Mollnow<sup>a</sup>, Kendra Croak<sup>a</sup>, Sarah Kane<sup>a</sup>, Kaitlyn Wagner<sup>a</sup>, Steven Dow<sup>a,d</sup> and Mark D. Zabel<sup>a</sup>

CONTACT Mark D. Zabel mzabel@colostate.edu

## ABSTRACT

Prion diseases are members of neurodegenerative, protein misfolding diseases (NPMDs) that include Alzheimer's, Parkinson's and Huntington diseases, Amyotrophic Lateral Sclerosis, tauopathies, traumatic brain injuries, and chronic traumatic encephalopathies. No known therapeutics improve quality of life or survival times of humans afflicted with prion disease. We and others developed a new approach to NPMD therapy based on reducing the amount of the normal, host-encoded protein that acts as a substrate for misfolding into pathologic forms using RNA interference, a pathway that decreases levels of mRNA encoding a particular protein. We developed a non-invasive therapeutic delivery system consisting of siRNA complexed to liposomes and addressed to the central nervous system using a targeting peptide derived from Rabies virus glycoprotein and delivered transvascularly. These liposome-siRNA-peptide complexes (LSPCs) cross the blood-brain barrier and deliver PrP siRNA to neuronal cells to decrease expression of the normal cellular prion

protein, PrPC, which acts as a substrate for prion replication. Here we show that LSPCs can extend survival and improve behaviour of prion-infected mice that remain immunotolerant to treatment. LSPC treatment may be a viable therapy for prion and other NPMDs that can improve the quality of life of patients at terminal disease stages.

## 246. Congenital hydrocephalus with drainage problems leading to CJD and Abeta accumulation. A case report

Annemieke Rozemuller, Wim Spliet, Anja Horstman, Will Hermsen, Vandana Gupta and Wim van Hecke

Prionlab. Dept. of Pathology, University Medical Center, Utrecht, The Netherlands

CONTACT Annemieke Rozemuller J.M.Rozemuller@umcutrecht.nl

Creutzfeldt-Jakob *amyloid beta *hydrocephalus *drainage.

## ABSTRACT

**Introduction:** We describe the neuropathology of a 48-year-old man with congenital hydrocephalus and CSF drain from his 18th year who became slow and got rapidly progressive dementia in 2 months, with epilepsy, dysarthria, gait abnormalities and myoclonus. The EEG was typical for CJD. The RT-QuIC test was positive.

**M&M:** The brain autopsy was performed in the prion-lab UMCUtrecht. The brain weight was 1410 g; the drain was situated in the bottom of the fourth ventricle. No hydrocephalus was seen but the aquaduct was extremely small. Twenty-five blocks were taken from relevant areas and stained for HE, Kluver-PAS as well immunohistochemically for 3F4. Next to that we stained for Abeta and tau.

**Results:** Routine staining showed fine vacuolar spongiosis of the grey matter and prion staining fitted with a histotype of MM/MV1 according to the histotyping of Parchi et al. (2012). In addition numerous diffuse Abeta plaques were found in the cortex but no classical amyloid plaques or (pre)tangles in the tau staining. Just a few dystrophic neurites.

**Conclusion:** This case report shows that congenital hydrocephalus with drainage problems can lead to protein misfolding with both prion accumulation (Creutzfeldt - Jakob disease) and numerous Abeta plaques on the age of 48 year. This supports earlier theories that drainage is essential for removing misfolded proteins.

<footnote><sup>a</sup>Prion Research Center, Department of Microbiology, Immunology and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA;
<sup>b</sup>Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO, USA;
<sup>c</sup>Department of Veterinary Population Medicine, College of Veterinary Medicine, University of Minnesota, St. Paul, MN, USA;
<sup>d</sup>Center for Immune and Regenerative Medicine, Department of Clinical Sciences, Colorado State University, Fort Collins, CO, USA</footnote>
<figure><img src="image_141.png" /></figure>

indicates that it is possible to direct hunting trips for CWD control by extending hunting seasons in CWD-infected areas without additional monetary or non-monetary incentives.

**KEYWORDS:** Chronic wasting disease; recreation demand

## Reference

[1] Potapov, et al. PLoS One. 2016;11(3):e0151039.

## 245. PrPC knockdown by Liposome-siRNA-peptide complexes (LSPCs) prolongs survival and normal behaviour in prion-infected mice immunotolerant to treatment

Heather Bender<sup>a,b</sup>, Noelle Noyes<sup>a,c</sup>, Jessica L Annis<sup>a</sup>, Amanda Hitpas<sup>a</sup>, Luke Mollnow<sup>a</sup>, Kendra Croak<sup>a</sup>, Sarah Kane<sup>a</sup>, Kaitlyn Wagner<sup>a</sup>, Steven Dow<sup>a,d</sup> and Mark D. Zabel<sup>a</sup>

CONTACT Mark D. Zabel mzabel@colostate.edu

## ABSTRACT

Prion diseases are members of neurodegenerative, protein misfolding diseases (NPMDs) that include Alzheimer's, Parkinson's and Huntington diseases, Amyotrophic Lateral Sclerosis, tauopathies, traumatic brain injuries, and chronic traumatic encephalopathies. No known therapeutics improve quality of life or survival times of humans afflicted with prion disease. We and others developed a new approach to NPMD therapy based on reducing the amount of the normal, host-encoded protein that acts as a substrate for misfolding into pathologic forms using RNA interference, a pathway that decreases levels of mRNA encoding a particular protein. We developed a non-invasive therapeutic delivery system consisting of siRNA complexed to liposomes and addressed to the central nervous system using a targeting peptide derived from Rabies virus glycoprotein and delivered transvascularly. These liposome-siRNA-peptide complexes (LSPCs) cross the blood-brain barrier and deliver PrP siRNA to neuronal cells to decrease expression of the normal cellular prion

protein, PrPC, which acts as a substrate for prion replication. Here we show that LSPCs can extend survival and improve behaviour of prion-infected mice that remain immunotolerant to treatment. LSPC treatment may be a viable therapy for prion and other NPMDs that can improve the quality of life of patients at terminal disease stages.

## 246. Congenital hydrocephalus with drainage problems leading to CJD and Abeta accumulation. A case report

Annemieke Rozemuller, Wim Spliet, Anja Horstman, Will Hermsen, Vandana Gupta and Wim van Hecke

Prionlab. Dept. of Pathology, University Medical Center, Utrecht, The Netherlands

CONTACT Annemieke Rozemuller J.M.Rozemuller@umcutrecht.nl

Creutzfeldt-Jakob *amyloid beta *hydrocephalus *drainage.

## ABSTRACT

**Introduction:** We describe the neuropathology of a 48-year-old man with congenital hydrocephalus and CSF drain from his 18th year who became slow and got rapidly progressive dementia in 2 months, with epilepsy, dysarthria, gait abnormalities and myoclonus. The EEG was typical for CJD. The RT-QuIC test was positive.

**M&M:** The brain autopsy was performed in the prion-lab UMCUtrecht. The brain weight was 1410 g; the drain was situated in the bottom of the fourth ventricle. No hydrocephalus was seen but the aquaduct was extremely small. Twenty-five blocks were taken from relevant areas and stained for HE, Kluver-PAS as well immunohistochemically for 3F4. Next to that we stained for Abeta and tau.

**Results:** Routine staining showed fine vacuolar spongiosis of the grey matter and prion staining fitted with a histotype of MM/MV1 according to the histotyping of Parchi et al. (2012). In addition numerous diffuse Abeta plaques were found in the cortex but no classical amyloid plaques or (pre)tangles in the tau staining. Just a few dystrophic neurites.

**Conclusion:** This case report shows that congenital hydrocephalus with drainage problems can lead to protein misfolding with both prion accumulation (Creutzfeldt - Jakob disease) and numerous Abeta plaques on the age of 48 year. This supports earlier theories that drainage is essential for removing misfolded proteins.

<footnote><sup>a</sup>Prion Research Center, Department of Microbiology, Immunology and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA;
<sup>b</sup>Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO, USA;
<sup>c</sup>Department of Veterinary Population Medicine, College of Veterinary Medicine, University of Minnesota, St. Paul, MN, USA;
<sup>d</sup>Center for Immune and Regenerative Medicine, Department of Clinical Sciences, Colorado State University, Fort Collins, CO, USA</footnote>
<figure><img src="image_138.png" /></figure>

## 247. Use of modern population genetic techniques to investigate the effects of the epidemic of kuru, a unique prion disease in the Papua New Guinea highlands

Liam Quinnª, Jerome Whitfieldª, Garrett Hellenthalᵇ, Michael Alpersᶜ,ᵈ, John Collingeª, and Simon Meadª

ªMRC Prion Unit at UCL, UCL Institute of Prion Diseases, London, UK; ᵇDepartment Genetics, Evolution & Environment, University College London; ᶜCentre for International Health, Curtin University, Perth, Australia; ᵈPapua New Guinea Institute of Medical Research, Goroka, Papua New Guinea

CONTACT Liam Quinn l.quinn@prion.ucl.ac.uk; Jerome Whitfield j.whitfield@prion.ucl.ac.uk

### ABSTRACT

**Background:** Kuru was the first observed and to this day remains the largest human prion disease epidemic. It devastatingly affected the Fore linguistic and nearby groups in the Papua New Guinea (PNG) highlands. The condition was transmitted by consumption of prion infected material at mortuary rituals. Over the first 20 documented years of the epidemic ~2,400 people died from kuru in communities with populations of ~40,000 individuals. The Eastern Highlands of Papua New Guinea where the Fore speaking communities reside is marked for its cultural and linguistic diversity. During the kuru epidemic the region was undergoing a marked transition with European contact and colonial administration. These factors present challenges to further understanding the population response to kuru specifically and approaches to find novel variants that affected disease predisposition.

**Materials and Methods:** We analysed a unique genome wide array dataset of 1,513 individuals from the affected communities from 22 linguistic groups and 50 across the Eastern Highlands of PNG. We used population genetic techniques including Chromopaintor analysis and fineStructure clustering to understand the population structure of the region, analyse patterns of gene flow and migration, tested for evidence of historical admixture events and evidence of genetic signatures of the impact of kuru on the Fore region including the marked sex bias in cases observed during the epidemic.

**Results:** We find a strong population structure with Principal Component Analysis and fineStructure clustering showing a strong tendency for individuals to cluster with individuals from the same linguistic grouping. Mean FST values observed in the Eastern Highlands were 0.02, far greater than those observed in the United Kingdom (0.0007) reflecting the degree of distinction between groups. Although we find that linguistic group membership explains many aspects of population structure we also

find that topographical features and cultural differences also exert strong forces. Gene flow between linguistic groups is evident with the presence of individuals reflecting historical processes of short and long distance migration.

**Conclusion:** We show that the affected populations have displayed remarkable resilience with no perceptible derivation in population genetic parameters overall despite facing such an existential threat. The marked population structure in the region and processes of gene flow present challenges for ongoing investigations into the genetic response and genetic architecture of kuru disease.

**KEYWORDS:** Kuru; population genetics; natural selection; genetic epidemiology

## 248. How the PrP<sup>C</sup> C-Terminal, globular domain regulates its toxic N-Terminal Domain

Glenn L. Millhauserª, David A. Harrisᵇ, Graham Rosemanª, Kevin Schillingª, Kate Markhamª, Alex McDonaldᵇ and Bei Wuᵇ

ªDepartment of Chemistry & Biochemistry, UC Santa Cruz, Santa Cruz, CA, USA; ᵇDepartment of Biochemistry, Boston University School of Medicine, Boston, MA, USA

CONTACT Glenn L. Millhauser glennm@ucsc.edu

### ABSTRACT

The cellular prion protein (PrP<sup>C</sup>) is composed of two primary segments – a helical C-terminal domain and a flexible N-terminal domain. Research over the last 5 years finds that the N-terminal domain is responsible for the monomeric protein's neuronal toxicity as revealed by toxic, spontaneous, electrophysiological currents and antibody induced neuronal death in cultured cells and brain slices. This inherent toxicity is arrested, however, by the regulatory C-terminal domain. Our research is focused on revealing the molecular mechanism underlying this critical regulatory function. The physiologic metal ions copper and zinc both bind to the octapeptide repeat segments in the N-terminal domain. Using advanced magnetic resonance approaches, along with electrophysiology measurements, we have identified a previously unseen intramolecular switch in PrP<sup>C</sup> whereby copper or zinc drive a tertiary contact between the N-terminal domain and a negatively charged, highly conserved surface on the C-terminal domain. Consistent with this model, we find that amino acid alterations at the docking surface that disrupt metal ion binding lead to toxic transmembrane currents. Together, our research provides a new paradigm for understanding PrP<sup>C</sup> toxicity and how familial mutations cause prion disease.
<figure><img src="image_142.png" /></figure>

## 247. Use of modern population genetic techniques to investigate the effects of the epidemic of kuru, a unique prion disease in the Papua New Guinea highlands

Liam Quinnª, Jerome Whitfieldª, Garrett Hellenthalᵇ, Michael Alpersᶜ,ᵈ, John Collingeª, and Simon Meadª

ªMRC Prion Unit at UCL, UCL Institute of Prion Diseases, London, UK; ᵇDepartment Genetics, Evolution & Environment, University College London; ᶜCentre for International Health, Curtin University, Perth, Australia; ᵈPapua New Guinea Institute of Medical Research, Goroka, Papua New Guinea

CONTACT Liam Quinn l.quinn@prion.ucl.ac.uk; Jerome Whitfield j.whitfield@prion.ucl.ac.uk

### ABSTRACT

**Background:** Kuru was the first observed and to this day remains the largest human prion disease epidemic. It devastatingly affected the Fore linguistic and nearby groups in the Papua New Guinea (PNG) highlands. The condition was transmitted by consumption of prion infected material at mortuary rituals. Over the first 20 documented years of the epidemic ~2,400 people died from kuru in communities with populations of ~40,000 individuals. The Eastern Highlands of Papua New Guinea where the Fore speaking communities reside is marked for its cultural and linguistic diversity. During the kuru epidemic the region was undergoing a marked transition with European contact and colonial administration. These factors present challenges to further understanding the population response to kuru specifically and approaches to find novel variants that affected disease predisposition.

**Materials and Methods:** We analysed a unique genome wide array dataset of 1,513 individuals from the affected communities from 22 linguistic groups and 50 across the Eastern Highlands of PNG. We used population genetic techniques including Chromopaint er analysis and fineStructure clustering to understand the population structure of the region, analyse patterns of gene flow and migration, tested for evidence of historical admixture events and evidence of genetic signatures of the impact of kuru on the Fore region including the marked sex bias in cases observed during the epidemic.

**Results:** We find a strong population structure with Principal Component Analysis and fineStructure clustering showing a strong tendency for individuals to cluster with individuals from the same linguistic grouping. Mean FST values observed in the Eastern Highlands were 0.02, far greater than those observed in the United Kingdom (0.0007) reflecting the degree of distinction between groups. Although we find that linguistic group membership explains many aspects of population structure we also

find that topographical features and cultural differences also exert strong forces. Gene flow between linguistic groups is evident with the presence of individuals reflecting historical processes of short and long distance migration.

**Conclusion:** We show that the affected populations have displayed remarkable resilience with no perceptible derivation in population genetic parameters overall despite facing such an existential threat. The marked population structure in the region and processes of gene flow present challenges for ongoing investigations into the genetic response and genetic architecture of kuru disease.

**KEYWORDS:** Kuru; population genetics; natural selection; genetic epidemiology

## 248. How the PrP<sup>C</sup> C-Terminal, globular domain regulates its toxic N-Terminal Domain

Glenn L. Millhauserª, David A. Harrisᵇ, Graham Rosemanª, Kevin Schillingª, Kate Markhamª, Alex McDonaldᵇ and Bei Wuᵇ

ªDepartment of Chemistry & Biochemistry, UC Santa Cruz, Santa Cruz, CA, USA; ᵇDepartment of Biochemistry, Boston University School of Medicine, Boston, MA, USA

CONTACT Glenn L. Millhauser glennm@ucsc.edu

### ABSTRACT

The cellular prion protein (PrP<sup>C</sup>) is composed of two primary segments – a helical C-terminal domain and a flexible N-terminal domain. Research over the last 5 years finds that the N-terminal domain is responsible for the monomeric protein's neuronal toxicity as revealed by toxic, spontaneous, electrophysiological currents and antibody induced neuronal death in cultured cells and brain slices. This inherent toxicity is arrested, however, by the regulatory C-terminal domain. Our research is focused on revealing the molecular mechanism underlying this critical regulatory function. The physiologic metal ions copper and zinc both bind to the octapeptide repeat segments in the N-terminal domain. Using advanced magnetic resonance approaches, along with electrophysiology measurements, we have identified a previously unseen intramolecular switch in PrP<sup>C</sup> whereby copper or zinc drive a tertiary contact between the N-terminal domain and a negatively charged, highly conserved surface on the C-terminal domain. Consistent with this model, we find that amino acid alterations at the docking surface that disrupt metal ion binding lead to toxic transmembrane currents. Together, our research provides a new paradigm for understanding PrP<sup>C</sup> toxicity and how familial mutations cause prion disease.
<figure><img src="image_139.png" /></figure>

## Funding

Supported by NIH grants R01 GM06790 (GM), S10 OD018455 (GM) and R01 NS065244 (DH).

## References

[1] Spevacek, et al. Structure. 2013;21:236-246.

[2] Evans, et al. Structure. 2016;24:1057-1067.

[3] Wu, et al. eLife. 2017. DOI:10.7554/eLife.23473.001

## 249. Validation studies of bovine heparin manufacturing process spiked with BSE agent

Cyrus Bett, Omozusi Andrews, Teresa Pilant, David M. Asher and Luisa Gregori

FDA/CBER/OBRR/DETTD/LBTSEA, Silver Spring, MD, USA

CONTACT Cyrus Bett Cyrus.bett@fda.hhs.gov

## ABSTRACT

Heparin is an injectable anticoagulant marketed in the US since the 1930's and used by 12 million Americans every year. Heparin is usually processed from mucosa of porcine or bovine intestines. In 2000, US manufacturers, concerned about possible contamination of bovine tissues with bovine spongiform encephalopathy (BSE) agent, stopped manufacturing bovine heparin. Thus, only porcine heparin is now marketed in the US, and about 75% originates from a single foreign country. Alternative sources of crude heparin would alleviate ongoing concerns about relying on single country and single animal species as a source of all heparin. This would also mitigate the risk of potential future shortages and episodes of adulteration, observed in the past. Bovine heparin is an obvious option, but BSE agent clearance by the bovine heparin manufacturing process should be evaluated. To this end, we applied a four-step bench-scale heparin purification protocol resembling a typical heparin manufacturing process to investigate removal of the spiked BSE agent. We removed aliquots from each step and analysed them for residual abnormal prion protein (PrP<sup>TSE</sup>) using real-time quaking-induced conversion (RT-QuIC) assay, and for infectivity using animal bioassays. The purification process reduced infectivity and PrP<sup>TSE</sup> by more than 3 logs. These findings show that the heparin manufacturing process has the capacity to remove substantial amounts of the BSE agent and support efforts to reintroduce safe bovine heparin in the USA.

## 250. Interaction with phosphatidic acid leads to prion protein fibrilization

R. F. D. Santos<sup>a,c</sup>, C. Alves<sup>a,b</sup>, L. V. Menezes<sup>b,c</sup>, J. M. A. Brito<sup>a,c</sup>, J. L. Silva<sup>a,c</sup> and T. C. R. G. Vieira<sup>a,c</sup>

<sup>a</sup>Medical Biochemistry Institute Leopoldo De Meis, Federal University of Rio de Janeiro; <sup>b</sup>Federal Institute of Rio de Janeiro; <sup>c</sup>National Institute of Cience and Tecnology of Estructural Biology and Bioimaging, Brazil

## ABSTRACT

Transmissible Spongiform Encephalopathies (TSEs) are neurodegenerative diseases caused by misfolding of the prion protein (PrP), which assumes a pathogenic conformation and propagates the infection through the conversion of native proteins. Recent models indicate the possible role of biological ligands in this process, which would act as cofactors in the conversion reaction. Lipids present in the mammalian cell membrane, especially in brain cells, have been suggested as endogenous adjuvants promoting prion aggregation. Despite this, little is known about the physical and chemical characteristics of this interaction. This study investigates the interaction and structural changes of recombinant PrP (murine, hamster and rabbit) in the presence of Phosphatidic Acid (PA) lipid vesicles, in an *in vitro* model. Full length or 90-231 fragment of rPrP<sup>C</sup> were incubated with PA vesicles. The samples were then analysed by spectroscopic techniques to detect changes in aggregation and tertiary structure. Increasing concentrations of PA raised rPrP light scattering, followed by changes in tryptophan fluorescence, indicating the formation of large aggregates. Interaction with PA induced an increase in beta-sheet content, with an intermediate enrichment compared to PrP<sup>Sc</sup>. These aggregates had an amorphous and amyloid fibre structure, shown by thioflavin T binding and microscopy observation. PrP truncated form showed more tendencies to form fibrils. rabPrP:PA aggregation was less robust than the other constructs and pH dependent. The results indicate that PA vesicles interact with rPrP, leading to the formation of amorphous aggregates and amyloid fibrils, close to PrP<sup>Sc</sup> secondary structure. The absence of the N-terminal is important to follow a fibrillization pathway induced by PA. rabPrP is able to interact with the biological cofactor tested, but with less effect depending on pH, suggesting that the different binding sites exposed at these pHs can be important.

**KEYWORDS:** Prion; neurodegenerative diseases; lipids; aggregation; vesicles
<figure><img src="image_143.png" /></figure>

## Funding

Supported by NIH grants R01 GM06790 (GM), S10 OD018455 (GM) and R01 NS065244 (DH).

## References

[1] Spevacek, et al. Structure. 2013;21:236-246.

[2] Evans, et al. Structure. 2016;24:1057-1067.

[3] Wu, et al. eLife. 2017. DOI:10.7554/eLife.23473.001

## 249. Validation studies of bovine heparin manufacturing process spiked with BSE agent

Cyrus Bett, Omozusi Andrews, Teresa Pilant, David M. Asher and Luisa Gregori

FDA/CBER/OBRR/DETTD/LBTSEA, Silver Spring, MD, USA

CONTACT Cyrus Bett Cyrus.bett@fda.hhs.gov

## ABSTRACT

Heparin is an injectable anticoagulant marketed in the US since the 1930's and used by 12 million Americans every year. Heparin is usually processed from mucosa of porcine or bovine intestines. In 2000, US manufacturers, concerned about possible contamination of bovine tissues with bovine spongiform encephalopathy (BSE) agent, stopped manufacturing bovine heparin. Thus, only porcine heparin is now marketed in the US, and about 75% originates from a single foreign country. Alternative sources of crude heparin would alleviate ongoing concerns about relying on single country and single animal species as a source of all heparin. This would also mitigate the risk of potential future shortages and episodes of adulteration, observed in the past. Bovine heparin is an obvious option, but BSE agent clearance by the bovine heparin manufacturing process should be evaluated. To this end, we applied a four-step bench-scale heparin purification protocol resembling a typical heparin manufacturing process to investigate removal of the spiked BSE agent. We removed aliquots from each step and analysed them for residual abnormal prion protein (PrP<sup>TSE</sup>) using real-time quaking-induced conversion (RT-QuIC) assay, and for infectivity using animal bioassays. The purification process reduced infectivity and PrP<sup>TSE</sup> by more than 3 logs. These findings show that the heparin manufacturing process has the capacity to remove substantial amounts of the BSE agent and support efforts to reintroduce safe bovine heparin in the USA.

## 250. Interaction with phosphatidic acid leads to prion protein fibrilization

R. F. D. Santos<sup>a,c</sup>, C. Alves<sup>a,b</sup>, L. V. Menezes<sup>b,c</sup>, J. M. A. Brito<sup>a,c</sup>, J. L. Silva<sup>a,c</sup> and T. C. R. G. Vieira<sup>a,c</sup>

<sup>a</sup>Medical Biochemistry Institute Leopoldo De Meis, Federal University of Rio de Janeiro; <sup>b</sup>Federal Institute of Rio de Janeiro; <sup>c</sup>National Institute of Cience and Tecnology of Estructural Biology and Bioimaging, Brazil

## ABSTRACT

Transmissible Spongiform Encephalopathies (TSEs) are neurodegenerative diseases caused by misfolding of the prion protein (PrP), which assumes a pathogenic conformation and propagates the infection through the conversion of native proteins. Recent models indicate the possible role of biological ligands in this process, which would act as cofactors in the conversion reaction. Lipids present in the mammalian cell membrane, especially in brain cells, have been suggested as endogenous adjuvants promoting prion aggregation. Despite this, little is known about the physical and chemical characteristics of this interaction. This study investigates the interaction and structural changes of recombinant PrP (murine, hamster and rabbit) in the presence of Phosphatidic Acid (PA) lipid vesicles, in an *in vitro* model. Full length or 90-231 fragment of rPrP<sup>C</sup> were incubated with PA vesicles. The samples were then analysed by spectroscopic techniques to detect changes in aggregation and tertiary structure. Increasing concentrations of PA raised rPrP light scattering, followed by changes in tryptophan fluorescence, indicating the formation of large aggregates. Interaction with PA induced an increase in beta-sheet content, with an intermediate enrichment compared to PrP<sup>Sc</sup>. These aggregates had an amorphous and amyloid fibre structure, shown by thioflavin T binding and microscopy observation. PrP truncated form showed more tendencies to form fibrils. rabPrP:PA aggregation was less robust than the other constructs and pH dependent. The results indicate that PA vesicles interact with rPrP, leading to the formation of amorphous aggregates and amyloid fibrils, close to PrP<sup>Sc</sup> secondary structure. The absence of the N-terminal is important to follow a fibrillization pathway induced by PA. rabPrP is able to interact with the biological cofactor tested, but with less effect depending on pH, suggesting that the different binding sites exposed at these pHs can be important.

**KEYWORDS:** Prion; neurodegenerative diseases; lipids; aggregation; vesicles
<figure><img src="image_140.png" /></figure>

**251. The photodynamic inactivation reduces prion infectivity in phthalocyanine treated RML brain homogenate**

Marie Kostelanska<sup>a</sup>, Zdenka Backovska Hanusova<sup>a</sup>, Jakub Soukup<sup>a</sup>, Radoslav Matej<sup>b</sup>, Karel Holada<sup>a</sup>

CONTACT Marie Kostelanska marie.kostelanska@lf1.cuni.cz

**ABSTRACT**

**Introduction:** Prions are highly resistant to conventional sterilization procedures and currently recommended inactivation methods include treatment with highly corrosive chemicals like 2% NaClO or 1M NaOH. Such harsh treatment is incompatible with a number of delicate medical tools. We have previously demonstrated that nontoxic disulfonated hydroxyaluminum phthalocyanine (AlPcOH(SO₃)₂) can be used to initiate photodynamic inactivation (PDI) of prions at ambient pressure and temperature.¹ The aim of this study was to demonstrate the effectiveness of PDI of prions by bioassay in laboratory mouse.

**Methods:** The PDI was performed on 1% RML or uninfectious CD1 brain homogenates (BH) treated with 25 µg ml⁻¹ of AlPcOH(SO₃)₂ and exposed to red light (18 min, 4 ×2.5W LED). Control RML inoculum was prepared similarly but was kept in the dark. In mouse bioassay, CD1 female mice (n = 10) were intracerebrally inoculated with a 25-µl aliquot of AlPcOH (SO₃)₂-light treated RML BH or control RML BH containing AlPcOH(SO₃)₂ but not irradiated by light. Other controls included mice (n = 5) inoculated with untreated, uninfectious CD1 BH or CD1 homogenate containing AlPcOH(SO₃)₂, either irradiated or nonirradiated. For comparison groups of mice (n = 5) were inoculated with 10-fold dilutions of RML BH (10⁻² to 10⁻⁶). The animals were sacrificed after reaching the terminal disease phase. The presence of PrPres in mouse brains was detected by WB and evaluation of PrPres distribution in brains by immunohistochemistry is ongoing.

**Results:** Mice inoculated with PDI-treated RML BH (at a 10⁻² dilution) survived significantly longer (204 ± 23 days) than mice inoculated with the homogenate containing an identical amount of AlPcOH(SO₃)₂, that was not exposed to light (165 ± 11 days). Their survival was also significantly longer than that of mice

inoculated with straight 10⁻², 10⁻³, and 10⁻⁴ dilutions of the RML BH, who survived 169 ± 11, 165 ± 12 and 176 ± 15 days, respectively. The transmission rate was 100% in all groups of mice including serial dilution (10⁻² to 10⁻⁶). A comparison of survival time-based on the regression line suggested a decrease in the prion infectivity titer of the RML BH of more than 4 orders of magnitude after PDI treatment.

**Conclusions:** Our results suggest that PDI with AlPcOH(SO₃)₂ leads to substantial decrease (~4 log₁₀) of prion infectivity in RML BH. The PDI of prions by light induced photocatalytic activity of phthalocyanines represents promising way of prion decontamination.

**KEYWORDS:** Prion; phthalocyanine; photodynamic inactivation; decontamination; mouse bioassay

**Funding**

The project was supported by AZV NV18-04-00179.

**Reference**

[1] Janouskova, et al. J Gen Virol. 2012;93(Pt11): 2512-7.

**252. RT-QuIC as an antemortem diagnostic tool to detect chronic wasting disease in deer skin**

Natália C. Ferreira<sup>a</sup>, Jorge M. Charco<sup>b</sup>, Michael A. Metrick<sup>a</sup>, Christina D. Orru<sup>a</sup>, Andrew G. Hughson<sup>a</sup>, Joaquín Castilla<sup>a</sup>, Michael W. Miller<sup>c</sup> and Byron Caughey<sup>a</sup>

CONTACT Natália C. Ferreira natalia.docarmoferreiradearaujo@nih.gov

**ABSTRACT**

Chronic wasting disease (CWD) is a fatal prion disease which affects cervids. This disease has an asymptomatic incubation time of 2-4 years, and during this period they can shed prions through saliva, feces, urine and placental tissue, contaminating the environment. Indeed, CWD highly contagious between cervids and shedding from live, infected animals likely contributes to its rapid spread. So far, CWD has been reported at least in 24 states in the US, as well as Canada, South Korea and Norway. To date, there is no evidence of CWD transmission to humans. However, in infected areas, deer population can drop as much as ~25 percent. Currently, there

<footnote><sup>a</sup>Institute of Immunology and Microbiology, First Faculty of Medicine, Charles University and General University Hospital in Prague, Czech Republic; <sup>b</sup>Department of Pathology and Molecular Medicine, Thomayer Teaching Hospital, Prague, Czech Republic</footnote>

<footnote><sup>a</sup>Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT, USA; <sup>2</sup> CIC bioGUNE, Derio (Bizkaia), Spain; <sup>3</sup>Colorado Division of Parks and Wildlife, Wildlife Health Program, Fort Collins, Colorado, USA</footnote>
<figure><img src="image_144.png" /></figure>

**251. The photodynamic inactivation reduces prion infectivity in phthalocyanine treated RML brain homogenate**

Marie Kostelanska<sup>a</sup>, Zdenka Backovska Hanusova<sup>a</sup>, Jakub Soukup<sup>a</sup>, Radoslav Matej<sup>b</sup>, Karel Holada<sup>a</sup>

CONTACT Marie Kostelanska marie.kostelanska@lf1.cuni.cz

**ABSTRACT**

**Introduction:** Prions are highly resistant to conventional sterilization procedures and currently recommended inactivation methods include treatment with highly corrosive chemicals like 2% NaClO or 1M NaOH. Such harsh treatment is incompatible with a number of delicate medical tools. We have previously demonstrated that nontoxic disulfonated hydroxyaluminum phthalocyanine (AlPcOH(SO₃)₂) can be used to initiate photodynamic inactivation (PDI) of prions at ambient pressure and temperature.¹ The aim of this study was to demonstrate the effectiveness of PDI of prions by bioassay in laboratory mouse.

**Methods:** The PDI was performed on 1% RML or uninfectious CD1 brain homogenates (BH) treated with 25 µg ml⁻¹ of AlPcOH(SO₃)₂ and exposed to red light (18 min, 4 ×2.5W LED). Control RML inoculum was prepared similarly but was kept in the dark. In mouse bioassay, CD1 female mice (n = 10) were intracerebrally inoculated with a 25-µl aliquot of AlPcOH (SO₃)₂-light treated RML BH or control RML BH containing AlPcOH(SO₃)₂ but not irradiated by light. Other controls included mice (n = 5) inoculated with untreated, uninfectious CD1 BH or CD1 homogenate containing AlPcOH(SO₃)₂, either irradiated or nonirradiated. For comparison groups of mice (n = 5) were inoculated with 10-fold dilutions of RML BH (10⁻² to 10⁻⁶). The animals were sacrificed after reaching the terminal disease phase. The presence of PrPres in mouse brains was detected by WB and evaluation of PrPres distribution in brains by immunohistochemistry is ongoing.

**Results:** Mice inoculated with PDI-treated RML BH (at a 10⁻² dilution) survived significantly longer (204 ± 23 days) than mice inoculated with the homogenate containing an identical amount of AlPcOH(SO₃)₂, that was not exposed to light (165 ± 11 days). Their survival was also significantly longer than that of mice

inoculated with straight 10⁻², 10⁻³, and 10⁻⁴ dilutions of the RML BH, who survived 169 ± 11, 165 ± 12 and 176 ± 15 days, respectively. The transmission rate was 100% in all groups of mice including serial dilution (10⁻² to 10⁻⁶). A comparison of survival time-based on the regression line suggested a decrease in the prion infectivity titer of the RML BH of more than 4 orders of magnitude after PDI treatment.

**Conclusions:** Our results suggest that PDI with AlPcOH(SO₃)₂ leads to substantial decrease (~4 log₁₀) of prion infectivity in RML BH. The PDI of prions by light induced photocatalytic activity of phthalocyanines represents promising way of prion decontamination.

**KEYWORDS:** Prion; phthalocyanine; photodynamic inactivation; decontamination; mouse bioassay

**Funding**

The project was supported by AZV NV18-04-00179.

**Reference**

[1] Janouskova, et al. J Gen Virol. 2012;93(Pt11): 2512-7.

**252. RT-QuIC as an antemortem diagnostic tool to detect chronic wasting disease in deer skin**

Natália C. Ferreira<sup>a</sup>, Jorge M. Charco<sup>b</sup>, Michael A. Metrick<sup>a</sup>, Christina D. Orru<sup>a</sup>, Andrew G. Hughson<sup>a</sup>, Joaquín Castilla<sup>a</sup>, Michael W. Miller<sup>c</sup> and Byron Caughey<sup>a</sup>

CONTACT Natália C. Ferreira natalia.docarmoferreiradearaujo@nih.gov

**ABSTRACT**

Chronic wasting disease (CWD) is a fatal prion disease which affects cervids. This disease has an asymptomatic incubation time of 2-4 years, and during this period they can shed prions through saliva, feces, urine and placental tissue, contaminating the environment. Indeed, CWD highly contagious between cervids and shedding from live, infected animals likely contributes to its rapid spread. So far, CWD has been reported at least in 24 states in the US, as well as Canada, South Korea and Norway. To date, there is no evidence of CWD transmission to humans. However, in infected areas, deer population can drop as much as ~25 percent. Currently, there

<footnote><sup>a</sup>Institute of Immunology and Microbiology, First Faculty of Medicine, Charles University and General University Hospital in Prague, Czech Republic; <sup>b</sup>Department of Pathology and Molecular Medicine, Thomayer Teaching Hospital, Prague, Czech Republic</footnote>

<footnote><sup>a</sup>Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT, USA; <sup>2</sup> CIC bioGUNE, Derio (Bizkaia), Spain; <sup>3</sup>Colorado Division of Parks and Wildlife, Wildlife Health Program, Fort Collins, Colorado, USA</footnote>
<figure><img src="image_141.png" /></figure>

are two tests approved to diagnose CWD: immuno-histochemistry and ELISA. However, these tests are applied *postmortem* and tissue types approved are the medial retropharyngeal lymph nodes and a specific region of brainstem (obex). Here we describe our efforts to adapt the real time quaking-induced conversion (RT-QuIC) as a diagnostic tool to detect $PrP^{CWD}$ in deer ear skin. Our initial analysis of a blinded panel of 50 samples yielded 82% sensitivity and 75% specificity. We are working to improve the conditions and performance of this assay, given that it might be useful for *antemortem* CWD diagnostics and surveillance.

## 253. Structural bioinformatics study on mechanisms of PrPc conformational stabilization mediated by the β 2- α 2 loop

Patricia Sotoª, Frances Mordenᵇ.c, India Claflinᶜ and Alyssa Bursottᵇ.c

ªPhysics department; ᵇNeuroscience program; ᶜBiology department, Creighton University, Omaha, NE, USA

### ABSTRACT

Understanding the factors that stabilize the globular C-terminal of the cellular form of the prion protein is key to decipher $PrP^C$ to $PrP^{Sc}$ conversion. The mechanism of this process is not well understood; however, studies suggest several factors affect the likelihood for prion infection. To understand the conformational stability of $PrP^C$, we examined the role of residue mutations, dimerization, and gpi-anchoring using structural bioinformatics techniques. Using structural bioinformatics techniques, we examined the role of point mutations and dimerization in the varying susceptibilities of $PrP^C$ structures. Our study on known resistant sheep $PrP^C$ structures indicates that key gene polymorphisms alter the network of residue interactions that involve the β 2- α 2 loop. This lowered susceptibility to initial protein misfolding likely occurs by rendering higher structural stability than the susceptible form. Our examination of prion protein dimerization suggests that the hydrophobic interactions at the dimer interface keep the dimer stable in the bulk and on the cell surface. The rotational mobility of dimers with respect to the cell surface is modulated by the gpi-anchoring state of the $PrP^C$ protein. With a comprehensive view of our results, we propose that if prion protein fibrillogenesis occurs following the nucleation-polymerization model, then key polymorphisms raise the energetic barrier to prevent templated prion protein conversion and dimerization conceals recognition spots that otherwise would be recognized by $PrP^{Sc}$ to bind and initiate prion protein conversion.

## 254. Humanized recombinant antibody fragments as tools for structural analyses of BSE and other prions

Vineet Rathodª,ᵇ, Jimmy Luª,ᵇ, José M Flores-Fernándezª,ᵇ, Xinli Tangª,ᵇ, Razieh Kamali-Jamilª,ᵇ, Leonardo Cortezª,ᶜ, Assunta Senatoreᵈ, Simone Hornemannᵈ, Valerie Simª,ᶜ, Adriano Aguzziᵈ and Holger Willeª,ᵇ

ªCenter for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Canada; ᵇDepartment of Biochemistry, University of Alberta, Edmonton, Canada; ᶜDepartment of Medicine, University of Alberta, Edmonton, Canada; ᵈInstitute of Neuropathology, University of Zürich, Zürich, Switzerland

### ABSTRACT

Prion diseases are rare, inexorably progressive, and fatal neurodegenerative disorders, with no therapy other than palliation. Prion diseases are unique compared in that they are transmissible, whereby the infectious agent ($PrP^{Sc}$) is propagated through the reproduction of its secondary, tertiary, and quaternary structure [1]. $PrP^{Sc}$ is a conformationally altered isoform of the cellular prion protein, $PrP^C$, which is a normal cell surface glycoprotein. Prion diseases affect humans (e.g. Creutzfeldt-Jakob disease: CJD), as well as a variety of animals, including chronic wasting disease (CWD) in deer and elk, scrapie in sheep and goats. Bovine Spongiform Encephalopathy (BSE) is an acquired transmissible prion disease in cattle, colloquially named ‘mad cow’ disease. It was first described in 1986 in the UK and has raised serious concerns about its transmission to humans, where it is known as variant CJD through consumption of contaminated meat [2]. While the structure of recombinant $PrP^C$ is extensively studied using various biophysical techniques, no high-resolution structure of $PrP^{Sc}$ has been published, and only its overall architecture as a four-rung β-solenoid is known [3,4].

Fab fragments (Fabs) are small antibody derivatives that maintain antigen-binding capacity, and can be used to study the structure of their antigens. One of the advantages of Fabs over intact antibodies is their small size, which allows them to penetrate deeper into protein aggregates, such as amyloid fibrils. We are using recombinant Fabs as an alternate approach in deciphering the structure of $PrP^{Sc}$ via immunogold labelling, electron microscopy, and difference mapping. One of our Fab library targets the four main regions of PrP: the charged cluster 1, octapeptide repeats, charged cluster 2, and globular domain. A second set of Fabs was recombinantly engineered from monoclonal antibodies (mAbs) that specifically recognize native $PrP^{Sc}$. These new $PrP^{Sc}$-specific mAbs (IgGs and IgMs) were recently generated in the Wille lab using a rationally designed, structure-based mimic for $PrP^{Sc}$.
<figure><img src="image_145.png" /></figure>

are two tests approved to diagnose CWD: immuno-
histochemistry and ELISA. However, these tests are
applied *postmortem* and tissue types approved are the
medial retropharyngeal lymph nodes and a specific
region of brainstem (obex). Here we describe our
efforts to adapt the real time quaking-induced con-
version (RT-QuIC) as a diagnostic tool to detect
PrP<sup>CWD</sup> in deer ear skin. Our initial analysis of a
blinded panel of 50 samples yielded 82% sensitivity
and 75% specificity. We are working to improve the
conditions and performance of this assay, given that
it might be useful for *antemortem* CWD diagnostics
and surveillance.

**253. Structural bioinformatics study on mechanisms of PrPc conformational stabilization mediated by the β 2- α 2 loop**

Patricia Sotoᵃ, Frances Mordenᵇ.c, India Claflinᶜ and Alyssa Bursottᵇ.c

ᵃPhysics department; ᵇNeuroscience program; ᶜBiology department, Creighton University, Omaha, NE, USA

ABSTRACT

Understanding the factors that stabilize the globular C-
terminal of the cellular form of the prion protein is key to
decipher PrP<sup>C</sup> to PrP<sup>Sc</sup> conversion. The mechanism of this
process is not well understood; however, studies suggest
several factors affect the likelihood for prion infection. To
understand the conformational stability of PrP<sup>C</sup>, we exam-
ined the role of residue mutations, dimerization, and gpi-
anchoring using structural bioinformatics techniques.
Using structural bioinformatics techniques, we examined
the role of point mutations and dimerization in the varying
susceptibilities of PrP<sup>C</sup> structures. Our study on known
resistant sheep PrP<sup>C</sup> structures indicates that key gene
polymorphisms alter the network of residue interactions
that involve the β 2- α 2 loop. This lowered susceptibility to
initial protein misfolding likely occurs by rendering higher
structural stability than the susceptible form. Our examina-
tion of prion protein dimerization suggests that the hydro-
phobic interactions at the dimer interface keep the dimer
stable in the bulk and on the cell surface. The rotational
mobility of dimers with respect to the cell surface is modu-
lated by the gpi-anchoring state of the PrP<sup>C</sup> protein. With a
comprehensive view of our results, we propose that if prion
protein fibrillogenesis occurs following the nucleation-
polymerization model, then key polymorphisms raise the
energetic barrier to prevent templated prion protein con-
version and dimerization conceals recognition spots that
otherwise would be recognized by PrP<sup>Sc</sup> to bind and initiate
prion protein conversion.

**254. Humanized recombinant antibody fragments as tools for structural analyses of BSE and other prions**

Vineet Rathod<sup>a,b</sup>, Jimmy Lu<sup>a,b</sup>, José M Flores-Fernández<sup>a,b</sup>, Xinli Tang<sup>a,b</sup>, Razieh Kamali-Jamil<sup>a,b</sup>, Leonardo Cortez<sup>a,c</sup>, Assunta Senatore<sup>d</sup>, Simone Hornemann<sup>d</sup>, Valerie Sim<sup>a,c</sup>, Adriano Aguzzi<sup>d</sup> and Holger Wille<sup>a,b</sup>

<sup>a</sup>Center for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Canada; <sup>b</sup>Department of Biochemistry, University of Alberta, Edmonton, Canada; <sup>c</sup>Department of Medicine, University of Alberta, Edmonton, Canada; <sup>d</sup>Institute of Neuropathology, University of Zürich, Zürich, Switzerland

ABSTRACT

Prion diseases are rare, inexorably progressive, and fatal
neurodegenerative disorders, with no therapy other than
palliation. Prion diseases are unique compared in that they
are transmissible, whereby the infectious agent (PrP<sup>Sc</sup>) is
propagated through the reproduction of its secondary,
tertiary, and quaternary structure [1]. PrP<sup>Sc</sup> is a conforma-
tionally altered isoform of the cellular prion protein, PrP<sup>C</sup>,
which is a normal cell surface glycoprotein. Prion diseases
affect humans (e.g. Creutzfeldt-Jakob disease: CJD), as well
as a variety of animals, including chronic wasting disease
(CWD) in deer and elk, scrapie in sheep and goats. Bovine
Spongiform Encephalopathy (BSE) is an acquired trans-
missible prion disease in cattle, colloquially named ‘mad
cow’ disease. It was first described in 1986 in the UK and
has raised serious concerns about its transmission to
humans, where it is known as variant CJD through con-
sumption of contaminated meat [2]. While the structure of
recombinant PrP<sup>C</sup> is extensively studied using various
biophysical techniques, no high-resolution structure of
PrP<sup>Sc</sup> has been published, and only its overall architecture
as a four-rung β-solenoid is known [3,4].

Fab fragments (Fabs) are small antibody derivatives that maintain antigen-binding capacity, and can be used to study the structure of their antigens. One of the advantages of Fabs over intact antibodies is their small size, which allows them to penetrate deeper into protein aggregates, such as amyloid fibrils. We are using recombinant Fabs as an alternate approach in deciphering the structure of PrP<sup>Sc</sup> via immunogold labelling, electron microscopy, and difference mapping. One of our Fab library targets the four main regions of PrP: the charged cluster 1, octapeptide repeats, charged cluster 2, and globular domain. A second set of Fabs was recombinantly engineered from monoclonal antibodies (mAbs) that specifically recognize native PrP<sup>Sc</sup>. These new PrP<sup>Sc</sup>-specific mAbs (IgGs and IgMs) were recently generated in the Wille lab using a rationally designed, structure-based mimic for PrP<sup>Sc</sup>.
<figure><img src="image_142.png" /></figure>

The main goal is to use *E. coli* to express fully func-
tional Fabs from both Fab libraries. Functionality assays
conducted after the expression and purification of the
Fabs indicate that some of them are able to bind native
PrP<sup>Sc</sup> fibrils. Complexes between Fabs and purified BSE
prion fibrils are being analysed using immunogold label-
ling and electron microscopy. Electron micrographs of
suitable prion fibrils will be used to generate 3D recon-
structions of labelled and unlabelled BSE fibrils and
provide insights into the orientation of the respective
epitopes in the β-solenoid architecture of PrP<sup>Sc</sup>.

**References**

[1] Wille H., Requena J. The structure of PrPSc prions. Pathogens. 2018;7(1):20.

[2] Aguzzi A., Sigurdson C.J. Antiprion immunotherapy: to suppress or to stimulate? Nat Rev Immunol. 2004;4 (9):725-736.

[3] Requena J.R., Wille H. The structure of the infectious prion protein and its propagation. Prog Mol Biol Transl Sci. 2017;150:341-359.

[4] Vazquez-Fernandez E., et al. The structural architecture of an infectious mammalian prion using electron cryomicroscopy. PLoS Pathog. 2016;12(9):e1005835.
<figure><img src="image_146.png" /></figure>

The main goal is to use *E. coli* to express fully func-
tional Fabs from both Fab libraries. Functionality assays
conducted after the expression and purification of the
Fabs indicate that some of them are able to bind native
PrP<sup>Sc</sup> fibrils. Complexes between Fabs and purified BSE
prion fibrils are being analysed using immunogold label-
ling and electron microscopy. Electron micrographs of
suitable prion fibrils will be used to generate 3D recon-
structions of labelled and unlabelled BSE fibrils and
provide insights into the orientation of the respective
epitopes in the β-solenoid architecture of PrP<sup>Sc</sup>.

**References**

[1] Wille H., Requena J. The structure of PrPSc prions. Pathogens. 2018;7(1):20.

[2] Aguzzi A., Sigurdson C.J. Antiprion immunotherapy: to suppress or to stimulate? Nat Rev Immunol. 2004;4 (9):725-736.

[3] Requena J.R., Wille H. The structure of the infectious prion protein and its propagation. Prog Mol Biol Transl Sci. 2017;150:341-359.

[4] Vazquez-Fernandez E., et al. The structural architecture of an infectious mammalian prion using electron cryomicroscopy. PLoS Pathog. 2016;12(9):e1005835.