<figure><img src="image_92.png" /></figure>

tg-elk$^{+/+}$ mice resulted in a single neuropathological profile that differed from the profile produced by passage of elk prions (described as the CWD2 strain [1]). Our results show that, upon first passage, the E226Q polymorphism did not affect the strain properties of deer prions and indicates a single strain (Wisc-1) was selected by the tg-elk$^{+/+}$ mice. The comparative analysis of neuropathological profiles between high and low expression tg-elk on first and second passage will be presented.

## References

[1] Johnson CJ, Herbst A, Duque-Velasquez C, et al. Prion protein polymorphisms affect chronic wasting disease progression. PLoS ONE. 2011;6(3):e17450.

[2] Duque Velasquez C, Kim C, Herbst A, et al. Deer prion proteins modulate the emergence and adaptation of chronic wasting disease strains. J Virol. 2015;89 (24):12,362–12,373

[3] Herbst A, et al. Chronic wasting disease prion strain emergence and host range expansion. Emerg Infect Dis. 2017;23(9):1598–1600.

[4] Angers R, et al. Prion strain mutation determined by prion protein conformational compatibility and primary structure. Science. 2010;328, 5982, 1154–1158.

[5] Le Dur, et al. Divergent prion strain evolution driven by PrPC expression level in transgenic mice. Nat Commun. 2017;8:14,170.

[6] La Fauci et al. Passage of chronic wasting disease prion into transgenic mice expressing Rocky Mountain elk (Cervus elaphus nelsoni) PrP<sup>C</sup>. J Gen Virol. 2006;87: 3773–3780.

# 143. A role for molecular chaperones in the rate of Alzheimer's disease

Sarah C. Hales<sup>a</sup>, Hailey Pineau<sup>a,b</sup>, Grant Norman<sup>a,b</sup> and Valerie L. Sim<sup>a,b</sup>

CONTACT Sarah C. Hales shales@ualberta.ca

## ABSTRACT

Despite similar pathological findings upon post-mortem examination of Alzheimer Disease (AD) brains (i.e. β-amyloid plaques and neurofibrillary tangles), the rate at which patients progress through this disease is highly variable, ranging from less than 2 years in very rare cases, to an average of 10 years. Previously, studies seeking to understand heterogeneity in disease progression have primarily looked for differences in the hallmark proteins associated with

AD: amyloid-β (Aβ) and tau. In terms of Aβ, studies have found differences in Aβ plaque composition1 and peptide structure2 in more rapidly progressing forms of the disease. Furthermore, the presence of different tau ‘strains’ has been suggested to influence its ability to aggregate and spread.3 However, the structural changes in Aβ and tau have not shed light on why disease duration varies so dramatically between patients. Our research into fast and slow cases of AD has revealed an increase in Aβ42 in the more slowly progressing cohort of patients, a finding that was unexpected as Aβ42 is often thought to be the more toxic species of Aβ. This may indicate that other factors present in the cellular environment may drive the rate of disease over and above any differences in Aβ and tau. Given the established role for molecular chaperones in the regulation of proteostasis and mediation of protein aggregation, they are ideal candidates to investigate with regards to rate of AD progression.

To test the role of chaperones in rate of disease, we are measuring levels of DNAJA2, HSP70, and HSC70 in human brains from patients with fast versus slow AD. We are also manipulating the levels of chaperones to determine their effect on Aβ and tau aggregation in cell culture model systems (N2A cells containing the Swedish amyloid precursor protein mutation, HEK293 cells containing the tau P301L mutation, and HEK293 cells containing the repeat domain of tau). A summary of these interactions and correlations will be presented.

## References

[1] Drummond E, et al. Acta Neuropathol 2017; 133:933–954.

[2] Cohen ML et al. Brain 2015; 138:1009–1022.

[3] Kaufman SK, et al. Neuron 2016; 92:796–812.

# 144. Deer prion protein polymorphisms influence in vitro aggregation kinetics

Helen Y. Yao<sup>a</sup>, Leonardo M. Cortez<sup>a,b</sup>, José Miguel Flores-Fernández<sup>a,c</sup>, Holger Wille<sup>a,c</sup>, Debbie McKenzie<sup>a,d</sup> and Valerie L. Sim<sup>a,b</sup>

CONTACT Helen Y. Yao hengyue@ualberta.ca

<footnote><sup>a</sup>Centre for Prions and Protein Folding Diseases, University of Alberta; <sup>b</sup>Department of Medicine, Division of Neurology, Faculty of Medicine & Dentistry, University of Alberta</footnote>

<footnote><sup>a</sup>Centre for Prions and Protein Folding Diseases, University of Alberta; <sup>b</sup>Department of Medicine, Division of Neurology, Faculty of Medicine & Dentistry, University of Alberta; <sup>c</sup>Department of Biochemistry, Faculty of Medicine & Dentistry, University of Alberta; <sup>d</sup>Department of Biological Sciences, Faculty of Science, University of Alberta</footnote>
<figure><img src="image_92.png" /></figure>

tg-elk$^{+/+}$ mice resulted in a single neuropathological profile that differed from the profile produced by passage of elk prions (described as the CWD2 strain [1]). Our results show that, upon first passage, the E226Q polymorphism did not affect the strain properties of deer prions and indicates a single strain (Wisc-1) was selected by the tg-elk$^{+/+}$ mice. The comparative analysis of neuropathological profiles between high and low expression tg-elk on first and second passage will be presented.

## References

[1] Johnson CJ, Herbst A, Duque-Velasquez C, et al. Prion protein polymorphisms affect chronic wasting disease progression. PLoS ONE. 2011;6(3):e17450.

[2] Duque Velasquez C, Kim C, Herbst A, et al. Deer prion proteins modulate the emergence and adaptation of chronic wasting disease strains. J Virol. 2015;89 (24):12,362–12,373

[3] Herbst A, et al. Chronic wasting disease prion strain emergence and host range expansion. Emerg Infect Dis. 2017;23(9):1598–1600.

[4] Angers R, et al. Prion strain mutation determined by prion protein conformational compatibility and primary structure. Science. 2010;328, 5982, 1154–1158.

[5] Le Dur, et al. Divergent prion strain evolution driven by PrPC expression level in transgenic mice. Nat Commun. 2017;8:14,170.

[6] La Fauci et al. Passage of chronic wasting disease prion into transgenic mice expressing Rocky Mountain elk (Cervus elaphus nelsoni) PrP<sup>C</sup>. J Gen Virol. 2006;87: 3773–3780.

# 143. A role for molecular chaperones in the rate of Alzheimer's disease

Sarah C. Hales<sup>a</sup>, Hailey Pineau<sup>a,b</sup>, Grant Norman<sup>a,b</sup> and Valerie L. Sim<sup>a,b</sup>

CONTACT Sarah C. Hales shales@ualberta.ca

## ABSTRACT

Despite similar pathological findings upon post-mortem examination of Alzheimer Disease (AD) brains (i.e. β-amyloid plaques and neurofibrillary tangles), the rate at which patients progress through this disease is highly variable, ranging from less than 2 years in very rare cases, to an average of 10 years. Previously, studies seeking to understand heterogeneity in disease progression have primarily looked for differences in the hallmark proteins associated with

AD: amyloid-β (Aβ) and tau. In terms of Aβ, studies have found differences in Aβ plaque composition1 and peptide structure2 in more rapidly progressing forms of the disease. Furthermore, the presence of different tau ‘strains’ has been suggested to influence its ability to aggregate and spread.3 However, the structural changes in Aβ and tau have not shed light on why disease duration varies so dramatically between patients. Our research into fast and slow cases of AD has revealed an increase in Aβ42 in the more slowly progressing cohort of patients, a finding that was unexpected as Aβ42 is often thought to be the more toxic species of Aβ. This may indicate that other factors present in the cellular environment may drive the rate of disease over and above any differences in Aβ and tau. Given the established role for molecular chaperones in the regulation of proteostasis and mediation of protein aggregation, they are ideal candidates to investigate with regards to rate of AD progression.

To test the role of chaperones in rate of disease, we are measuring levels of DNAJA2, HSP70, and HSC70 in human brains from patients with fast versus slow AD. We are also manipulating the levels of chaperones to determine their effect on Aβ and tau aggregation in cell culture model systems (N2A cells containing the Swedish amyloid precursor protein mutation, HEK293 cells containing the tau P301L mutation, and HEK293 cells containing the repeat domain of tau). A summary of these interactions and correlations will be presented.

## References

[1] Drummond E, et al. Acta Neuropathol 2017; 133:933–954.

[2] Cohen ML et al. Brain 2015; 138:1009–1022.

[3] Kaufman SK, et al. Neuron 2016; 92:796–812.

# 144. Deer prion protein polymorphisms influence in vitro aggregation kinetics

Helen Y. Yao<sup>a</sup>, Leonardo M. Cortez<sup>a,b</sup>, José Miguel Flores-Fernández<sup>a,c</sup>, Holger Wille<sup>a,c</sup>, Debbie McKenzie<sup>a,d</sup> and Valerie L. Sim<sup>a,b</sup>

CONTACT Helen Y. Yao hengyue@ualberta.ca

<footnote><sup>a</sup>Centre for Prions and Protein Folding Diseases, University of Alberta; <sup>b</sup>Department of Medicine, Division of Neurology, Faculty of Medicine & Dentistry, University of Alberta</footnote>

<footnote><sup>a</sup>Centre for Prions and Protein Folding Diseases, University of Alberta; <sup>b</sup>Department of Medicine, Division of Neurology, Faculty of Medicine & Dentistry, University of Alberta; <sup>c</sup>Department of Biochemistry, Faculty of Medicine & Dentistry, University of Alberta; <sup>d</sup>Department of Biological Sciences, Faculty of Science, University of Alberta</footnote>
<figure><img src="image_93.png" /></figure>

**ABSTRACT**

**Introduction:** Chronic wasting disease (CWD) is a transmissible neurodegenerative disease of cervids. It is caused by the misfolding and self-templated aggregation of the prion protein (PrP). Different polymorphisms of PrP are associated with different *in vivo* disease susceptibility, as shown in mice, elk and white-tailed deer [1]. Polymorphisms such as 95H and 96S in deer Prnp are associated with altered transmission properties, but the mechanism by which this happens is not clear. Deer heterozygous for the *with G96S and/or Q95H polymorphisms* have longer incubation periods than deer expressing wt Prnp [1]. Mice expressing 96S deer PrP are not susceptible to Wisc-1 strain CWD. Interestingly, PrP aggregates containing 95H are able to infect mice expressing 96S [2]. If these transmission differences are caused by different aggregation and seeding efficiencies of the three polymorphic PrP types, we may find similar correlations with *in vitro* aggregation studies: PrP containing 96S or 95H should be less effective as a substrate for conversion, unless the seed contains 95H-PrP. Similarly, we expect any aggregate with 95H to be more efficient than 96S as a seed.

**Methods:** Wild type, 96S and 95H deer PrP were expressed in *E. coli* and purified from inclusion bodies by affinity chromatography followed by HPLC. Monomeric PrP was aggregated using real-time quaking induced conversion (RT-QuIC) to study the kinetics of spontaneous, homologously seeded and heterologously seeded aggregation. Lag phases and final ThT fluorescence values were compared.

**Results:** As predicted, 96S was the most inefficient substrate for conversion, even when homologously seeded. Spontaneously formed aggregates of 96S were also the least efficient seeds. 95H, on the other hand, produced aggregates that were very efficient at seeding, even more than wildtype aggregates. 95H was also an excellent substrate for conversion, being more efficient than wildtype.

**Conclusions:** The inefficient aggregation and seeding ability of 96S PrP correlates with its long incubation period *in vivo*, suggesting this amino acid change could explain the *in vivo* findings. Similarly, the high seeding ability of 95H PrP aggregates correlates with the finding that 95H strains can infect otherwise resistance mice (96S mice). However, 95H was a very efficient substrate *in vitro* which is not what has been observed in deer heterozygous for 95H; whether deer homozygous for 95H would have faster incubation periods remains to be seen.

**References**

[1] Johnson C, et al. J Gen Virol 2006;87:2109–114.

[2] Duque Velasquez C, et al. J Virol 2015;89:12,362–73.

**145. Deciphering ovine scrapie complexity in Europe by using bioassay in two rodent models**

Alba Marín-Morenoª, Patricia Aguilar-Calvoª, José Luis Pitarchª, Juan Carlos Espinosaª, Olivier Andreolettiᵇ, Romolo Nonnoᶜ, Jan Langeveldᵈ and Juan María Torresª

ªCentro de Investigación en Sanidad Animal (CISA-INIA), Madrid, Spain; ᵇÉcole Nationale Vétérinaire de Toulouse, Toulouse, France; ᶜIstituto Superiore di Sanità, Rome, Italy; ᵈWageningen BioVeterinary Research, Lelystad, the Netherlands.

**CONTACT** Alba Marín-Moreno marin.alba@inia.es

**ABSTRACT**

Within the prion field, scrapie tends to be named as a uniform disease caused by a single prion strain. However, as the opposite that applies for Bovine Spongiform Encephalopathy, several scrapie strains producing different prion diseases phenotypes have been reported in the literature. Previous work using European goat prion isolates already pointed to this situation (Nonno et al.). Goat and sheep scrapie isolates proceeding from several European countries were intracranially inoculated into transgenic mice overexpressing ovine and bovine PrP proteins (Ov-Tg501 and Bo-Tg110 respectively). Two iterative passages were performed. The combination of the two different rodent models allows the classification of the isolates in four groups that may correspond to several scrapie strains. Abrogation of the transmission barrier by performing the second passage is necessary to correctly classify the isolates. In addition, Bo-Tg110 provides the most important information to allow discrimination between groups.

**KEYWORDS:** Scrapie; prion strain; transmission barrier; Europe; mouse bioassay.

**Funding**

Spanish Ministerio de Ciencia, Innovación y Universidades (AGL2016-78054R) and European Union projects CT-2006-36,353 and 219,235 ERA-NET FP7 EMIDA.

**146. Conversion kinetics of sporadic CJD prions using multiple substrates and RT-QuIC-based detection and discrimination**

Stephanie Boothª, Sarah Medinaᵇ, Lise Lamoureuxᵇ and Debra Sorensenᶜ
<figure><img src="image_93.png" /></figure>

## ABSTRACT

**Introduction:** Chronic wasting disease (CWD) is a transmissible neurodegenerative disease of cervids. It is caused by the misfolding and self-templated aggregation of the prion protein (PrP). Different polymorphisms of PrP are associated with different *in vivo* disease susceptibility, as shown in mice, elk and white-tailed deer [1]. Polymorphisms such as 95H and 96S in deer Prnp are associated with altered transmission properties, but the mechanism by which this happens is not clear. Deer heterozygous for the *with G96S and/or Q95H polymorphisms* have longer incubation periods than deer expressing wt Prnp [1]. Mice expressing 96S deer PrP are not susceptible to Wisc-1 strain CWD. Interestingly, PrP aggregates containing 95H are able to infect mice expressing 96S [2]. If these transmission differences are caused by different aggregation and seeding efficiencies of the three polymorphic PrP types, we may find similar correlations with *in vitro* aggregation studies: PrP containing 96S or 95H should be less effective as a substrate for conversion, unless the seed contains 95H-PrP. Similarly, we expect any aggregate with 95H to be more efficient than 96S as a seed.

**Methods:** Wild type, 96S and 95H deer PrP were expressed in *E. coli* and purified from inclusion bodies by affinity chromatography followed by HPLC. Monomeric PrP was aggregated using real-time quaking induced conversion (RT-QuIC) to study the kinetics of spontaneous, homologously seeded and heterologously seeded aggregation. Lag phases and final ThT fluorescence values were compared.

**Results:** As predicted, 96S was the most inefficient substrate for conversion, even when homologously seeded. Spontaneously formed aggregates of 96S were also the least efficient seeds. 95H, on the other hand, produced aggregates that were very efficient at seeding, even more than wildtype aggregates. 95H was also an excellent substrate for conversion, being more efficient than wildtype.

**Conclusions:** The inefficient aggregation and seeding ability of 96S PrP correlates with its long incubation period *in vivo*, suggesting this amino acid change could explain the *in vivo* findings. Similarly, the high seeding ability of 95H PrP aggregates correlates with the finding that 95H strains can infect otherwise resistance mice (96S mice). However, 95H was a very efficient substrate *in vitro* which is not what has been observed in deer heterozygous for 95H; whether deer homozygous for 95H would have faster incubation periods remains to be seen.

## References

[1] Johnson C, et al. J Gen Virol 2006;87:2109-114.

[2] Duque Velasquez C, et al. J Virol 2015;89:12,362-73.

## 145. Deciphering ovine scrapie complexity in Europe by using bioassay in two rodent models

Alba Marín-Morenoª, Patricia Aguilar-Calvoª, José Luis Pitarchª, Juan Carlos Espinosaª, Olivier Andreolettiᵇ, Romolo Nonnoᶜ, Jan Langeveldᵈ and Juan María Torresª

ªCentro de Investigación en Sanidad Animal (CISA-INIA), Madrid, Spain; ᵇÉcole Nationale Vétérinaire de Toulouse, Toulouse, France; ᶜIstituto Superiore di Sanità, Rome, Italy; ᵈWageningen BioVeterinary Research, Lelystad, the Netherlands.

CONTACT Alba Marín-Moreno marin.alba@inia.es

## ABSTRACT

Within the prion field, scrapie tends to be named as a uniform disease caused by a single prion strain. However, as the opposite that applies for Bovine Spongiform Encephalopathy, several scrapie strains producing different prion diseases phenotypes have been reported in the literature. Previous work using European goat prion isolates already pointed to this situation (Nonno et al.). Goat and sheep scrapie isolates proceeding from several European countries were intracranially inoculated into transgenic mice overexpressing ovine and bovine PrP proteins (Ov-Tg501 and Bo-Tg110 respectively). Two iterative passages were performed. The combination of the two different rodent models allows the classification of the isolates in four groups that may correspond to several scrapie strains. Abrogation of the transmission barrier by performing the second passage is necessary to correctly classify the isolates. In addition, Bo-Tg110 provides the most important information to allow discrimination between groups.

**KEYWORDS:** Scrapie; prion strain; transmission barrier; Europe; mouse bioassay.

## Funding

Spanish Ministerio de Ciencia, Innovación y Universidades (AGL2016-78054R) and European Union projects CT-2006-36,353 and 219,235 ERA-NET FP7 EMIDA.

## 146. Conversion kinetics of sporadic CJD prions using multiple substrates and RT-QuIC-based detection and discrimination

Stephanie Boothª, Sarah Medinaᵇ, Lise Lamoureuxᵇ and Debra Sorensenᶜ
<figure><img src="image_94.png" /></figure>

ᵃPHAC; ᵇPublic Health Agency of Canada; ᶜPublic Health Agency of Canada-NML

## ABSTRACT

Prions often maintain their conformational characteristics upon natural transmission to a new species and that this is dependent on the structural characteristics of both the 'infecting' PrP and the new host PrP. Based on the hypothesis that variations in prion conformation are intrinsic to strain characteristics and ultimately disease phenotypes, it seems logical to conclude that the seed - substrate pairing has a direct relationship to the conversion rate in assays such as real-time quaking-induced conversion (RT-QuIC). We have used RT-QuIC to assess the relative rates of conversion of CJD prions from over 40 patients for whom CJD was confirmed as the cause of death by the Canadian CJD Surveillance System. These patients included those diagnosed with five subtypes of sporadic CJD, sCJDMM1/sCJDMV1, sCJDVV2, sCJDMV2, SCJDMM2 and sCJDVV1. A number of different recombinant prion proteins including hamster, elk and human were used as substrate. Serial dilution of brain tissue from these individuals were used as seeds. We hypothesized that the degree of complementarity during seed-substrate pairing would provide a measure of strain conformation based on the relative efficiency of the initial amyloid conversion of a particular substrate by a seed. We measured the conversion rates by comparing the time at which fluorescence in each reaction exceeded a pre-defined threshold, i.e. the lag phase. Lag phases were extremely consistent for each CJD type in the different substrates so that we could statistically compare cases and assign to different types. Of interest, we found a small number of atypical cases whose conversion kinetics differed significantly from others within their subtype perhaps indicating different strain types. We also performed seeding studies on sCJD prions isolated from specific brain regions of interest providing information on the heterogeneity of prions related to the brain region in which they are propagated.

## 147. Novel, pan-PrP<sup>Sc</sup>-specific antibodies reveal a shared β-solenoid structure for all PrP<sup>Sc</sup> strains tested

Holger Wille<sup>a,b</sup>, Andrew Fang<sup>a,b</sup>, Xinli Tang<sup>a,b</sup>, Madeleine Fleming<sup>a,b</sup>, Tolu Abodunwa<sup>a,b</sup>, Leonardo M. Cortez<sup>a,c</sup>, Claudia Y. Acevedo-Morantes<sup>a,b</sup>, Camilo Duque Velásquez<sup>a,d</sup>, Brian Tancowny<sup>a,b</sup>, Xiongyao Wang<sup>a,b,§</sup>, Judd Aiken<sup>a,e</sup>, Debbie McKenzie<sup>a,d</sup> and Valerie L. Sim<sup>a,c</sup>

CONTACT Holger Wille wille@ualberta.ca

<sup>§</sup>current address: School of Materials Science and Engineering, Harbin Institute of Technology, Weihai, Shandong, China.

## ABSTRACT

**Background:** The high-resolution structure of PrP<sup>Sc</sup> has eluded experimental determination due to its insolubility and its general propensity to aggregate. Nevertheless, the repeating nature of PrP<sup>Sc</sup> amyloid fibrils has allowed the collection of lower-resolution structural data via X-ray fibre diffraction, cryo-electron microscopy, and other techniques. All of these approaches indicated that the structure of PrP<sup>Sc</sup> is based on a four-rung, parallel β-solenoid architecture.

**Materials and Methods:** The aforementioned β-solenoid architecture allowed us to predict which amino acid side chains may be oriented towards the surface of PrP<sup>Sc</sup> versus those that form the interior of the β-solenoid fold. Based on these predictions, we created a structural and immunological mimic for PrP<sup>Sc</sup> by inserting outwards facing residues into an identical position in an innocuous protein scaffold based on the fungal prion HET-s. This structural and immunological mimic (=vaccine candidate) was then injected into wild-type mice and their immune response analysed. The resultant splenocytes were used to generate hybridoma cells for the production of monoclonal antibodies. These monoclonal antibodies were characterized for their ability to recognize native PrP<sup>Sc</sup>. Lastly, a systematic series of revertant mutants of the PrP<sup>Sc</sup>-based vaccine candidate towards the unmodified HET-s scaffold were used to further refine the epitopes that these antibodies recognize.

**Results:** Immunization with the structure-based vaccine candidate resulted in a PrP<sup>Sc</sup>-specific immune response in 8 out of 8 mice. The post-immune antiserum recognized only native PrP<sup>Sc</sup>, and did not bind to PrP<sup>C</sup>, recombinant PrP, denatured PrP, or any linear PrP peptides. The monoclonal antibodies that were derived from the immunized mice (IgGs and IgMs) recognized all natural PrP<sup>Sc</sup> strains/isolates, including BSE, CWD, TME, scrapie, and human prions (familial: fCJD, FFI, GSS; sporadic: sCJD; infectious: vCJD). Moreover, the monoclonal antibodies did not recognize *in vitro* generated recPrP amyloid, which presumably adopted a parallel in-register intermolecular β-sheet (PIRIBS) architecture. Lastly, the HET-s revertant mutants helped to define the PrP<sup>Sc</sup> epitopes within the framework of the HET-s β-solenoid structure.

**Conclusions:** Our novel, pan-PrP<sup>Sc</sup>-specific antibodies confirm the hypothesis that the structure of PrP<sup>Sc</sup> contains a parallel β-solenoid fold. In particular, these antibodies recognize structural epitopes that are

<footnote><sup>a</sup>Centre for Prions and Protein Folding Diseases, University of Alberta; <sup>b</sup>Department of Biochemistry; <sup>c</sup>Department of Medicine; <sup>d</sup>Department of Biological Sciences; <sup>e</sup>Department of Agricultural, Food, and Nutritional Science, Edmonton, Alberta, Canada.</footnote>
<figure><img src="image_94.png" /></figure>

ᵃPHAC; ᵇPublic Health Agency of Canada; ᶜPublic Health Agency of Canada-NML

## ABSTRACT

Prions often maintain their conformational characteristics upon natural transmission to a new species and that this is dependent on the structural characteristics of both the 'infecting' PrP and the new host PrP. Based on the hypothesis that variations in prion conformation are intrinsic to strain characteristics and ultimately disease phenotypes, it seems logical to conclude that the seed - substrate pairing has a direct relationship to the conversion rate in assays such as real-time quaking-induced conversion (RT-QuIC). We have used RT-QuIC to assess the relative rates of conversion of CJD prions from over 40 patients for whom CJD was confirmed as the cause of death by the Canadian CJD Surveillance System. These patients included those diagnosed with five subtypes of sporadic CJD, sCJDMM1/sCJDMV1, sCJDVV2, sCJDMV2, SCJDMM2 and sCJDVV1. A number of different recombinant prion proteins including hamster, elk and human were used as substrate. Serial dilution of brain tissue from these individuals were used as seeds. We hypothesized that the degree of complementarity during seed-substrate pairing would provide a measure of strain conformation based on the relative efficiency of the initial amyloid conversion of a particular substrate by a seed. We measured the conversion rates by comparing the time at which fluorescence in each reaction exceeded a pre-defined threshold, i.e. the lag phase. Lag phases were extremely consistent for each CJD type in the different substrates so that we could statistically compare cases and assign to different types. Of interest, we found a small number of atypical cases whose conversion kinetics differed significantly from others within their subtype perhaps indicating different strain types. We also performed seeding studies on sCJD prions isolated from specific brain regions of interest providing information on the heterogeneity of prions related to the brain region in which they are propagated.

# 147. Novel, pan-PrP<sup>Sc</sup>-specific antibodies reveal a shared β-solenoid structure for all PrP<sup>Sc</sup> strains tested

Holger Wille<sup>a,b</sup>, Andrew Fang<sup>a,b</sup>, Xinli Tang<sup>a,b</sup>, Madeleine Fleming<sup>a,b</sup>, Tolu Abodunwa<sup>a,b</sup>, Leonardo M. Cortez<sup>a,c</sup>, Claudia Y. Acevedo-Morantes<sup>a,b</sup>, Camilo Duque Velásquez<sup>a,d</sup>, Brian Tancowny<sup>a,b</sup>, Xiongyao Wang<sup>a,b,§</sup>, Judd Aiken<sup>a,e</sup>, Debbie McKenzie<sup>a,d</sup> and Valerie L. Sim<sup>a,c</sup>

CONTACT Holger Wille wille@ualberta.ca

<sup>s</sup>current address: School of Materials Science and Engineering, Harbin Institute of Technology, Weihai, Shandong, China.

## ABSTRACT

**Background:** The high-resolution structure of PrP<sup>Sc</sup> has eluded experimental determination due to its insolubility and its general propensity to aggregate. Nevertheless, the repeating nature of PrP<sup>Sc</sup> amyloid fibrils has allowed the collection of lower-resolution structural data via X-ray fibre diffraction, cryo-electron microscopy, and other techniques. All of these approaches indicated that the structure of PrP<sup>Sc</sup> is based on a four-rung, parallel β-solenoid architecture.

**Materials and Methods:** The aforementioned β-solenoid architecture allowed us to predict which amino acid side chains may be oriented towards the surface of PrP<sup>Sc</sup> versus those that form the interior of the β-solenoid fold. Based on these predictions, we created a structural and immunological mimic for PrP<sup>Sc</sup> by inserting outwards facing residues into an identical position in an innocuous protein scaffold based on the fungal prion HET-s. This structural and immunological mimic (=vaccine candidate) was then injected into wild-type mice and their immune response analysed. The resultant spleno-cytes were used to generate hybridoma cells for the production of monoclonal antibodies. These monoclonal antibodies were characterized for their ability to recognize native PrP<sup>Sc</sup>. Lastly, a systematic series of revertant mutants of the PrP<sup>Sc</sup>-based vaccine candidate towards the unmodified HET-s scaffold were used to further refine the epitopes that these antibodies recognize.

**Results:** Immunization with the structure-based vaccine candidate resulted in a PrP<sup>Sc</sup>-specific immune response in 8 out of 8 mice. The post-immune antiserum recognized only native PrP<sup>Sc</sup>, and did not bind to PrP<sup>C</sup>, recombinant PrP, denatured PrP, or any linear PrP peptides. The monoclonal antibodies that were derived from the immunized mice (IgGs and IgMs) recognized all natural PrP<sup>Sc</sup> strains/isolates, including BSE, CWD, TME, scrapie, and human prions (familial: fCJD, FFI, GSS; sporadic: sCJD; infectious: vCJD). Moreover, the monoclonal antibodies did not recognize *in vitro* generated recPrP amyloid, which presumably adopted a parallel in-register intermolecular β-sheet (PIRIBS) architecture. Lastly, the HET-s revertant mutants helped to define the PrP<sup>Sc</sup> epitopes within the framework of the HET-s β-solenoid structure.

**Conclusions:** Our novel, pan-PrP<sup>Sc</sup>-specific antibodies confirm the hypothesis that the structure of PrP<sup>Sc</sup> contains a parallel β-solenoid fold. In particular, these antibodies recognize structural epitopes that are

<footnote><sup>a</sup>Centre for Prions and Protein Folding Diseases, University of Alberta; <sup>b</sup>Department of Biochemistry; <sup>c</sup>Department of Medicine; <sup>d</sup>Department of Biological Sciences; <sup>e</sup>Department of Agricultural, Food, and Nutritional Science, Edmonton, Alberta, Canada.</footnote>
<figure><img src="image_95.png" /></figure>

specific for the parallel β-solenoid fold of PrP<sup>Sc</sup>, but do not recognize alternative conformers of PrP including β-sheet rich PIRIBS structures. These antibodies and the original antigen are currently being studied for their potential use as passive or active immunotherapy agents and for use in diagnostic applications.

**148. Characterization of infectious human prions**

Claudia Y. Acevedo-Morantes<sup>a,b</sup>, Xiongyao Wang<sup>a,b,*</sup>, Brian Tancowny<sup>a,b</sup>, Susana Teijeira<sup>c,d</sup>, Beatriz San Millán<sup>c,d</sup> and Holger Wille<sup>a,b</sup>

<sup>a</sup>Centre for Prions and Protein Folding Diseases; <sup>b</sup>Department of Biochemistry, University of Alberta, Edmonton, AB, Canada; <sup>c</sup>Galicia Sur Health Research Institute, IIS Galicia Sur, SERGAS-UVIGO, Vigo, Spain; <sup>d</sup>Pathology Department, Complexo Hospitalario Universitario de Vigo (CHUVI), IIS Galicia Sur, SERGAS, Vigo, Spain.

CONTACT Claudia Y. Acevedo-Morantes cacevedo@ualberta.ca

*Current address: School of Materials Science and Engineering, Harbin Institute of Technology, Weihai, Shandong, China.

ABSTRACT

Human prion diseases are a family of rare and progressive neurodegenerative disorders. They present as sporadic, familial, infectious, or iatrogenic forms, and include Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome (GSS), and Fatal Familial Insomnia (FFI). The causes for the wide range of phenotypic variation in the human prion diseases are unknown, but the disease is caused by aberrantly folded versions of the prion protein, termed PrPSc. Biochemical properties such as partial resistance to digestion by proteinase K, insolubility in non-ionic detergents, and sedimentation properties have provided evidence that distinct forms of PrPSc aggregates with heterogeneous morphologies regarding fibril length, width, shape, longitudinal twist, and the number of protofilaments that may characterize these disease entities. In this study, we are using several biochemical and biophysical approaches to analyse and compare PrPSc-fractions derived from the brains of patients affected by sporadic CJD (sCJD), familial CJD (fCJD), variant CJD (vCJD), GSS, and FFI. It is hypothesized that the structure of PrPSc is the key factor that determines its infectious nature, transmission route, and pathogenicity. However, the insolubility of PrPSc and its overall propensity to aggregate make it inaccessible to high-resolution structure analyses. Low-resolution approaches have provided information on the architecture of PrPSc to devise molecular models. Using tilted-beam transmission electron microscopy (TB-TEM), we are measuring the mass-per-length (MPL) value of individual PrPSc fibrils derived from the brains of patients

affected by the aforementioned prion diseases. These results will provide a platform to evaluate the proposed models for the structure of PrPSc fibrils. Tobacco Mosaic Virus (TMV), with its well-determined structure and an MPL value of 131 kDa/nm, serves as an internal calibration standard for the MPL measurements. Preliminary results obtained with purified PrPSc fibrils from sCJD samples indicated a population of fibrils with a MPL value around 60 kDa/nm, which would support the four-rung β-solenoid model based on two protofilaments per fibril. Additional MPL measurements on GSS patient-derived samples are underway. Recombinant PrP amyloid fibrils as well as *in vitro* converted recombinant PrPSc will be used to round out the MPL comparison of prion fibril structures. MPL data impose a stringent constraint on viable fibril models when the subunit mass is known. By calibrating the image intensity of PrPSc to the internal standard, we will be able to limit the number of possible indexing schemes significantly, and provide independent experimental validation to the current proposed models.

**149. Rationally designed, structure-based vaccines candidates for αβ, tau and α-synuclein**

José M Flores-Fernández, Enrique Chimal-Juárez, Xiongyao Wang*, Brian Tancowny, Xinli Tang, Andrew Fang and Holger Wille

Centre for Prions and Protein Folding Diseases & Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada.

CONTACT José M Flores-Fernández floresfe@ualberta.ca

*current address: School of Materials Science and Engineering, Harbin Institute of Technology, Weihai, Shandong, China.

ABSTRACT

**Background:** Alzheimer's and Parkinson's diseases are mainly caused by the misfolding of Aβ, tau, and α-synuclein proteins, respectively. Each of these proteins adopts a beta-sheet rich conformation and aggregates into amyloid. Different attempts to develop vaccines for these diseases have used the respective proteins and peptides in their linear form and without control of their tertiary and quaternary structure, which caused a lack of specificity and subsequently a failure to achieve protection. In this study, we present a new approach to design vaccine candidates based on structurally defined proteins that result in disease-specific antigenicity.

**Materials and Methods:** The fungal prion Het-s was used as an innocuous scaffold protein because it natively adopts a conformation rich in beta-sheet. This scaffold protein was engineered based on the surface exposed amino acids of Aβ, tau, and α-synuclein
<figure><img src="image_95.png" /></figure>

specific for the parallel β-solenoid fold of PrP<sup>Sc</sup>, but do not recognize alternative conformers of PrP including β-sheet rich PIRIBS structures. These antibodies and the original antigen are currently being studied for their potential use as passive or active immunotherapy agents and for use in diagnostic applications.

**148. Characterization of infectious human prions**

Claudia Y. Acevedo-Morantes<sup>a,b</sup>, Xiongyao Wang<sup>a,b,*</sup>, Brian Tancowny<sup>a,b</sup>, Susana Teijeira<sup>c,d</sup>, Beatriz San Millán<sup>c,d</sup> and Holger Wille<sup>a,b</sup>

<sup>a</sup>Centre for Prions and Protein Folding Diseases; <sup>b</sup>Department of Biochemistry, University of Alberta, Edmonton, AB, Canada; <sup>c</sup>Galicia Sur Health Research Institute, IIS Galicia Sur, SERGAS-UVIGO, Vigo, Spain; <sup>d</sup>Pathology Department, Complexo Hospitalario Universitario de Vigo (CHUVI), IIS Galicia Sur, SERGAS, Vigo, Spain.

CONTACT Claudia Y. Acevedo-Morantes cacevedo@ualberta.ca

*Current address: School of Materials Science and Engineering, Harbin Institute of Technology, Weihai, Shandong, China.

**ABSTRACT**

Human prion diseases are a family of rare and progressive neurodegenerative disorders. They present as sporadic, familial, infectious, or iatrogenic forms, and include Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome (GSS), and Fatal Familial Insomnia (FFI). The causes for the wide range of phenotypic variation in the human prion diseases are unknown, but the disease is caused by aberrantly folded versions of the prion protein, termed PrPSc. Biochemical properties such as partial resistance to digestion by proteinase K, insolubility in non-ionic detergents, and sedimentation properties have provided evidence that distinct forms of PrPSc aggregates with heterogeneous morphologies regarding fibril length, width, shape, longitudinal twist, and the number of protofilaments that may characterize these disease entities. In this study, we are using several biochemical and biophysical approaches to analyse and compare PrPSc-fractions derived from the brains of patients affected by sporadic CJD (sCJD), familial CJD (fCJD), variant CJD (vCJD), GSS, and FFI. It is hypothesized that the structure of PrPSc is the key factor that determines its infectious nature, transmission route, and pathogenicity. However, the insolubility of PrPSc and its overall propensity to aggregate make it inaccessible to high-resolution structure analyses. Low-resolution approaches have provided information on the architecture of PrPSc to devise molecular models. Using tilted-beam transmission electron microscopy (TB-TEM), we are measuring the mass-per-length (MPL) value of individual PrPSc fibrils derived from the brains of patients

affected by the aforementioned prion diseases. These results will provide a platform to evaluate the proposed models for the structure of PrPSc fibrils. Tobacco Mosaic Virus (TMV), with its well-determined structure and an MPL value of 131 kDa/nm, serves as an internal calibration standard for the MPL measurements. Preliminary results obtained with purified PrPSc fibrils from sCJD samples indicated a population of fibrils with a MPL value around 60 kDa/nm, which would support the four-rung β-solenoid model based on two protofilaments per fibril. Additional MPL measurements on GSS patient-derived samples are underway. Recombinant PrP amyloid fibrils as well as *in vitro* converted recombinant PrPSc will be used to round out the MPL comparison of prion fibril structures. MPL data impose a stringent constraint on viable fibril models when the subunit mass is known. By calibrating the image intensity of PrPSc to the internal standard, we will be able to limit the number of possible indexing schemes significantly, and provide independent experimental validation to the current proposed models.

**149. Rationally designed, structure-based vaccines candidates for αβ, tau and α-synuclein**

José M Flores-Fernández, Enrique Chimal-Juárez, Xiongyao Wang*, Brian Tancowny, Xinli Tang, Andrew Fang and Holger Wille

Centre for Prions and Protein Folding Diseases & Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada.

CONTACT José M Flores-Fernández floresfe@ualberta.ca

*current address: School of Materials Science and Engineering, Harbin Institute of Technology, Weihai, Shandong, China.

**ABSTRACT**

**Background:** Alzheimer's and Parkinson's diseases are mainly caused by the misfolding of Aβ, tau, and α-synuclein proteins, respectively. Each of these proteins adopts a beta-sheet rich conformation and aggregates into amyloid. Different attempts to develop vaccines for these diseases have used the respective proteins and peptides in their linear form and without control of their tertiary and quaternary structure, which caused a lack of specificity and subsequently a failure to achieve protection. In this study, we present a new approach to design vaccine candidates based on structurally defined proteins that result in disease-specific antigenicity.

**Materials and Methods:** The fungal prion Het-s was used as an innocuous scaffold protein because it natively adopts a conformation rich in beta-sheet. This scaffold protein was engineered based on the surface exposed amino acids of Aβ, tau, and α-synuclein
<figure><img src="image_96.png" /></figure>

aggregates to express antigenic determinants in a struc-
turally controlled manner. The resulting vaccine candi-
dates were produced in *Escherichia coli*, purified,
refolded, and then analysed by EM for the proper
folding of the proteins. Only those constructs that
formed amyloid fibrils with the typical structure of
Het-s amyloid were injected into mice. The resulting
polyclonal antibodies were tested against unmodified
Het-s, the engineered Het-s antigen, and disease-speci-
fic antigens in order to determine their specificity.

**Results:** After the purification and refolding of the engineered proteins, several vaccine candidates that targeted Aβ, tau, and α-synuclein showed the expected self-assembly into amyloid fibrils by negative stain electron microscopy. The self-assembled constructs were injected in mice and the collected antisera were tested for their specificity by ELISAs using Het-s and its engineered variants as antigens. The vaccine candidates Aβ4 and Tau4 elicited immune responses with higher reactivity for the engineered antigens than for unmodified Het-s.

**Conclusions:** Rationally designed, structure-based vac-
cine candidates targeting the misfolded proteins
responsible for Alzheimer's and Parkinson's disease
are feasible with this approach.

150. Characterization of the glycosylation profile of water-soluble prion protein from brain and blood of hamster using lectins

Hanin Abdel-Haq

Istituto Superiore di Sanità, Rome, Italy

CONTACT Hanin Abdel-Haq Hanin.abdelhaq@iss.it

ABSTRACT

**Background:** The disease-associated water-soluble form of hamster prion protein (ws-PrP<sup>Sc</sup>) has recently been found to be less stable than classical PrP<sup>Sc</sup>. Since the stability of PrP to enzymatic degradation correlates with its glycosylation level, the aim of this study was to investigate whether there are differences between the glycosylation of ws-PrP<sup>Sc</sup> and classical PrP<sup>Sc</sup> of hamster.

**Materials and methods:** To achieve this aim, ws-PrP and classical PrP were captured from noninfected or scrapie-infected hamster brain homogenate [high-speed supernatant (S<sup>HS</sup>) and high-speed pellet (P<sup>HS</sup>)] and blood plasma by anti-PrP antibodies (3F4 and 6H4) and subjected to screening for glycans by seven biotinylated lectins under denaturing or nondenaturing conditions in a sandwich lectin enzyme-linked immunosorbent assay (lectin-ELISA).

**Results:** Glycans have been found in minor quantities and differently exposed on ws-PrP<sup>Sc</sup> from S<sup>HS</sup> and plasma compared with classical PrP<sup>Sc</sup> from P<sup>HS</sup>. These differences have been shown to be potentially responsible for the instability of ws-PrP<sup>Sc</sup>. Treatment of infected blood with guanidine hydrochloride (GdnHCl) significantly (P <0.01) increased the detection of ws-PrP<sup>Sc</sup> in ELISA, reflecting an increase in its stability, and showed efficacy in removing high-abundance proteins in silver-stained gels. This increase in ws-PrP<sup>Sc</sup> stability is due to an interaction of GdnHCl not only with high-abundance proteins but also with the ws-PrP<sup>Sc</sup> glycosylation with particular regard to the mannose sugar. Analysis of lectins immunoreactivity towards total proteins from plasma collected before and at different time points after infection, revealed that mannose might exert a stabilizing effect towards all of hamster blood glycoproteins, regardless of scrapie infection. Since low levels of ws-PrP<sup>Sc</sup>/soluble-infectivity have been estimated both in blood and brain of hamster, this glycosylation-related instability may have negatively influenced the propensity of ws-PrP<sup>C</sup> to convert to ws-PrP<sup>Sc</sup> both in blood and the brain.

**Conclusions:** Glycosylation characteristics of cellular prion protein (PrP<sup>C</sup>) may provide a tool for the determination risk of prion transmissibility among different species and tissues. These results could be representative for other misfolded proteins that are characterized by having different glycoforms, including the soluble form, such as amyloid beta in Alzheimer's disease.

**KEYWORDS:** Glycosylation; Prion protein; Guanidine-induced protein stabilization; Lectins; Blood; Misfolded proteins.

151. Early detection of prion protein aggregation with pFTAA using spectral confocal microscopy

Waqas Tahirª, Anastasiia Stepanchukᵇ, Hermann M Schatzlª and Peter K Stysᵇ

CONTACT Waqas Tahir waqas.tahir1@ucalgary.ca

ABSTRACT

**Background:** Prion diseases are fatal neurodegen-
erative diseases characterized by the formation of
amyloidogenic prion aggregates in the CNS. Prion
aggregates are formed by conformational alteration
of host encoded cellular prion protein into the

<footnote>ªDepartment of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, Alberta, Canada; ᵇDepartment of Clinical Neuroscience, Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta, Canada</footnote>
<figure><img src="image_96.png" /></figure>

aggregates to express antigenic determinants in a struc-
turally controlled manner. The resulting vaccine candi-
dates were produced in *Escherichia coli*, purified,
refolded, and then analysed by EM for the proper
folding of the proteins. Only those constructs that
formed amyloid fibrils with the typical structure of
Het-s amyloid were injected into mice. The resulting
polyclonal antibodies were tested against unmodified
Het-s, the engineered Het-s antigen, and disease-speci-
fic antigens in order to determine their specificity.

**Results:** After the purification and refolding of the engineered proteins, several vaccine candidates that targeted Aβ, tau, and α-synuclein showed the expected self-assembly into amyloid fibrils by negative stain electron microscopy. The self-assembled constructs were injected in mice and the collected antisera were tested for their specificity by ELISAs using Het-s and its engineered variants as antigens. The vaccine candidates Aβ4 and Tau4 elicited immune responses with higher reactivity for the engineered antigens than for unmodified Het-s.

**Conclusions:** Rationally designed, structure-based vac-
cine candidates targeting the misfolded proteins
responsible for Alzheimer's and Parkinson's disease
are feasible with this approach.

150. Characterization of the glycosylation profile of water-soluble prion protein from brain and blood of hamster using lectins

Hanin Abdel-Haq

Istituto Superiore di Sanità, Rome, Italy

CONTACT Hanin Abdel-Haq Hanin.abdelhaq@iss.it

ABSTRACT

**Background:** The disease-associated water-soluble form of hamster prion protein (ws-PrP<sup>Sc</sup>) has recently been found to be less stable than classical PrP<sup>Sc</sup>. Since the stability of PrP to enzymatic degradation correlates with its glycosylation level, the aim of this study was to investigate whether there are differences between the glycosylation of ws-PrP<sup>Sc</sup> and classical PrP<sup>Sc</sup> of hamster.

**Materials and methods:** To achieve this aim, ws-PrP and classical PrP were captured from noninfected or scrapie-infected hamster brain homogenate [high-speed supernatant (S<sup>HS</sup>) and high-speed pellet (P<sup>HS</sup>)] and blood plasma by anti-PrP antibodies (3F4 and 6H4) and subjected to screening for glycans by seven biotinylated lectins under denaturing or nondenaturing conditions in a sandwich lectin enzyme-linked immunosorbent assay (lectin-ELISA).

**Results:** Glycans have been found in minor quantities and differently exposed on ws-PrP<sup>Sc</sup> from S<sup>HS</sup> and plasma compared with classical PrP<sup>Sc</sup> from P<sup>HS</sup>. These differences have been shown to be potentially responsible for the instability of ws-PrP<sup>Sc</sup>. Treatment of infected blood with guanidine hydrochloride (GdnHCl) significantly (P <0.01) increased the detection of ws-PrP<sup>Sc</sup> in ELISA, reflecting an increase in its stability, and showed efficacy in removing high-abundance proteins in silver-stained gels. This increase in ws-PrP<sup>Sc</sup> stability is due to an interaction of GdnHCl not only with high-abundance proteins but also with the ws-PrP<sup>Sc</sup> glycosylation with particular regard to the mannose sugar. Analysis of lectins immunoreactivity towards total proteins from plasma collected before and at different time points after infection, revealed that mannose might exert a stabilizing effect towards all of hamster blood glycoproteins, regardless of scrapie infection. Since low levels of ws-PrP<sup>Sc</sup>/soluble-infectivity have been estimated both in blood and brain of hamster, this glycosylation-related instability may have negatively influenced the propensity of ws-PrP<sup>C</sup> to convert to ws-PrP<sup>Sc</sup> both in blood and the brain.

**Conclusions:** Glycosylation characteristics of cellular prion protein (PrP<sup>C</sup>) may provide a tool for the determination risk of prion transmissibility among different species and tissues. These results could be representative for other misfolded proteins that are characterized by having different glycoforms, including the soluble form, such as amyloid beta in Alzheimer's disease.

**KEYWORDS:** Glycosylation; Prion protein; Guanidine-induced protein stabilization; Lectins; Blood; Misfolded proteins.

151. Early detection of prion protein aggregation with pFTAA using spectral confocal microscopy

Waqas Tahirª, Anastasiia Stepanchukᵇ, Hermann M Schatzlª and Peter K Stysᵇ

CONTACT Waqas Tahir waqas.tahir1@ucalgary.ca

ABSTRACT

**Background:** Prion diseases are fatal neurodegen-
erative diseases characterized by the formation of
amyloidogenic prion aggregates in the CNS. Prion
aggregates are formed by conformational alteration
of host encoded cellular prion protein into the

<footnote>ªDepartment of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, Alberta, Canada; ᵇDepartment of Clinical Neuroscience, Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta, Canada</footnote>
<figure><img src="image_97.png" /></figure>

scrapie form. Quick progression in disease pathology after the onset of symptoms makes such diseases deadlier. Early detection of prion aggregates can help to better understand the pathophysiology and spread of prion infection in the brain. In this regard, amyloid sensitive fluorescent probes and advanced analytical tools to quantify emission spectra of those fluorescent probes can help to detect these pathological events at earlier stages of disease progression.

**Objective:** Early detection of protein aggregates in prion infected mouse brains by examining changes in emission spectrum of brain sections stained with the amyloid-sensitive fluorescent probe pFTAA by using spectral confocal microscopy.

**Materials and Methods:** FVB mice were intra-cerebrally inoculated with 20 µL of brain homogenates (mock or 22 L infected) from terminal mice. Prion infected and age matched mock inoculated mice were euthanized for pre-symptomatic stages (at 75 and 100 dpi), symptomatic stage (125 dpi) and terminal stage. Brain tissues were extracted and fixed with formalin. Brain sections (5 µm) were hydrated followed by incubation with pFTAA (3 µM) for 45 min. Stained sections were imaged with confocal microscope equipped with a 32-channel spectral detector. Image analysis was done in custom-written software ImageTrak using a non-linear spectral unmixing algorithm.

**Results:** Fluorescence spectral analysis of prion-infected brain sections from pre-symptomatic stages (75 and 100 dpi) showed increased fluorescence intensity as well as a characteristic red shift in the emission spectrum as compared to age matched mock brain sections. Differences in emission spectrum between prion infected and mock inoculated brain sections increased at later stages of the disease. Spectral differences at pre-symptomatic stages were mainly seen in somatosensory cortex, hippocampus and thalamus. Pathological alterations represented by changes in emission spectrum during symptomatic and terminal stages spread to further brain regions including olfactory bulb, hypothalamus, cerebellum, and brainstem.

**Conclusions:** Advanced spectral imaging analysis of pFTAA stained brain sections infected with 22 L prions showed robust differences in emission spectrum during prion infection. This shows the potential of such methods for early detection of prion aggregates, possibly also in the tissues other than only brain, and also can offer better understanding about mechanism of prion spread in protein misfolding disorders with higher sensitivity.

**152. Tissue tropism of prions is dictated by the balance between PrP<sup>C</sup> formation and degradation**

Ronald A. Shikiya<sup>a</sup>, Katie A. Langenfeld<sup>b</sup>, Thomas E. Eckland<sup>c</sup>, Jonathan Trinh<sup>a</sup>, Sara A. M. Holec<sup>a</sup>, Candace K. Mathiason<sup>d,e</sup>, Anthony E. Kincaid<sup>a,e</sup> and Jason C. Bartz<sup>a</sup>

<sup>a</sup>Department of Medical Microbiology and Immunology, Creighton University, Omaha, NE, USA; <sup>b</sup>Biology Department, University of Nebraska at Omaha, Omaha, NE, USA; <sup>c</sup>Department of Neurology, University of Texas Health Science Center, Houston, TX, USA; <sup>d</sup>Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO, USA; <sup>e</sup>Department of Pharmacy Science, Creighton University, Omaha, NE, USA

CONTACT Ronald A. Shikiya ronaldshikiya@creighton.edu

**ABSTRACT**

Prion strains are characterized by strain-specific differences in neuropathology, incubation period, clinical disease, host-range, and tissue tropism. In hamsters, the hyper (HY) and drowsy (DY) prion strains differ in tissue tropism and susceptibility to disease when extra-neural routes of infection are used. Notably, hamster susceptibility to the DY strain is restricted when inoculation occurs via extraneural routes. In contrast to the HY strain, DY is not detected in secondary lymphoreticular system (LRS) tissues of infected hosts regardless of the route of infection. Our experiments show that, similar to the lymphotropic strain HY, DY crosses the mucosal epithelia, enters the draining lymphatic vessels in underlying laminae propriae, and is transported to LRS tissues. Since DY is able to cause disease once it enters the peripheral nervous system, the restriction in DY pathogenesis is not due to a failure of transport but may be due to its inability to stablish infection in LRS tissues. To determine if DY can propagate in LRS tissues, we performed protein misfolding cyclic amplification using DY PrP<sup>Sc</sup> as the seed and spleen homogenate as the source of PrP<sup>C</sup>. Our results show that the spleen environment can support DY PrP<sup>Sc</sup> formation, albeit at lower levels when compared to other lymphotropic prion strains, suggesting that the restriction in DY pathogenesis is not due to the absence of a strain-specific co-factor in spleen. In addition, DY PrP<sup>Sc</sup> is more susceptible to degradation compared to PrP<sup>Sc</sup> from other lymphotropic strains. Based on these results, we suggest that the equilibrium between PrP<sup>Sc</sup> formation and clearance plays a role in prion tissue tropism.

**153. Molecular mechanisms of Aβ fibril elongation, secondary nucleation, and fibril dissociation studied in-silico**

Lyudmyla Dorosh<sup>a,b</sup> and Maria Stepanova<sup>b,c</sup>
<figure><img src="image_97.png" /></figure>

scrapie form. Quick progression in disease pathology after the onset of symptoms makes such diseases deadlier. Early detection of prion aggregates can help to better understand the pathophysiology and spread of prion infection in the brain. In this regard, amyloid sensitive fluorescent probes and advanced analytical tools to quantify emission spectra of those fluorescent probes can help to detect these pathological events at earlier stages of disease progression.

**Objective:** Early detection of protein aggregates in prion infected mouse brains by examining changes in emission spectrum of brain sections stained with the amyloid-sensitive fluorescent probe pFTAA by using spectral confocal microscopy.

**Materials and Methods:** FVB mice were intra-cerebrally inoculated with 20 µL of brain homogenates (mock or 22 L infected) from terminal mice. Prion infected and age matched mock inoculated mice were euthanized for pre-symptomatic stages (at 75 and 100 dpi), symptomatic stage (125 dpi) and terminal stage. Brain tissues were extracted and fixed with formalin. Brain sections (5 µm) were hydrated followed by incubation with pFTAA (3 µM) for 45 min. Stained sections were imaged with confocal microscope equipped with a 32-channel spectral detector. Image analysis was done in custom-written software ImageTrak using a non-linear spectral unmixing algorithm.

**Results:** Fluorescence spectral analysis of prion-infected brain sections from pre-symptomatic stages (75 and 100 dpi) showed increased fluorescence intensity as well as a characteristic red shift in the emission spectrum as compared to age matched mock brain sections. Differences in emission spectrum between prion infected and mock inoculated brain sections increased at later stages of the disease. Spectral differences at pre-symptomatic stages were mainly seen in somatosensory cortex, hippocampus and thalamus. Pathological alterations represented by changes in emission spectrum during symptomatic and terminal stages spread to further brain regions including olfactory bulb, hypothalamus, cerebellum, and brainstem.

**Conclusions:** Advanced spectral imaging analysis of pFTAA stained brain sections infected with 22 L prions showed robust differences in emission spectrum during prion infection. This shows the potential of such methods for early detection of prion aggregates, possibly also in the tissues other than only brain, and also can offer better understanding about mechanism of prion spread in protein misfolding disorders with higher sensitivity.

**152. Tissue tropism of prions is dictated by the balance between PrP<sup>Sc</sup> formation and degradation**

Ronald A. Shikiya<sup>a</sup>, Katie A. Langenfeld<sup>b</sup>, Thomas E. Eckland<sup>c</sup>, Jonathan Trinh<sup>a</sup>, Sara A. M. Holec<sup>a</sup>, Candace K. Mathiason<sup>d,e</sup>, Anthony E. Kincaid<sup>a,e</sup> and Jason C. Bartz<sup>a</sup>

<sup>a</sup>Department of Medical Microbiology and Immunology, Creighton University, Omaha, NE, USA; <sup>b</sup>Biology Department, University of Nebraska at Omaha, Omaha, NE, USA; <sup>c</sup>Department of Neurology, University of Texas Health Science Center, Houston, TX, USA; <sup>d</sup>Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO, USA; <sup>e</sup>Department of Pharmacy Science, Creighton University, Omaha, NE, USA

CONTACT Ronald A. Shikiya ronaldshikiya@creighton.edu

**ABSTRACT**

Prion strains are characterized by strain-specific differences in neuropathology, incubation period, clinical disease, host-range, and tissue tropism. In hamsters, the hyper (HY) and drowsy (DY) prion strains differ in tissue tropism and susceptibility to disease when extra-neural routes of infection are used. Notably, hamster susceptibility to the DY strain is restricted when inoculation occurs via extraneural routes. In contrast to the HY strain, DY is not detected in secondary lymphoreticular system (LRS) tissues of infected hosts regardless of the route of infection. Our experiments show that, similar to the lymphotropic strain HY, DY crosses the mucosal epithelia, enters the draining lymphatic vessels in underlying laminae propriae, and is transported to LRS tissues. Since DY is able to cause disease once it enters the peripheral nervous system, the restriction in DY pathogenesis is not due to a failure of transport but may be due to its inability to stablish infection in LRS tissues. To determine if DY can propagate in LRS tissues, we performed protein misfolding cyclic amplification using DY PrP<sup>Sc</sup> as the seed and spleen homogenate as the source of PrP<sup>C</sup>. Our results show that the spleen environment can support DY PrP<sup>Sc</sup> formation, albeit at lower levels when compared to other lymphotropic prion strains, suggesting that the restriction in DY pathogenesis is not due to the absence of a strain-specific co-factor in spleen. In addition, DY PrP<sup>Sc</sup> is more susceptible to degradation compared to PrP<sup>Sc</sup> from other lymphotropic strains. Based on these results, we suggest that the equilibrium between PrP<sup>Sc</sup> formation and clearance plays a role in prion tissue tropism.

**153. Molecular mechanisms of Aβ fibril elongation, secondary nucleation, and fibril dissociation studied in-silico**

Lyudmyla Dorosh<sup>a,b</sup> and Maria Stepanova<sup>b,c</sup>
<figure><img src="image_98.png" /></figure>

<sup>a</sup>Department of Electrical and Computer Engineering, University of Alberta, Edmonton, Canada; <sup>b</sup>National Institute for Nanotechnology NRC; Edmonton, Canada; <sup>c</sup>Department of Electrical and Computer Engineering, University of Alberta, Edmonton, Canada

CONTACT Lyudmyla Dorosh dorosh@ualberta.ca; Maria Stepanova ms1@ualberta.ca

## ABSTRACT

Prevalence in ageing population, difficulties in early diagnosis, and lack of efficient therapies make Alzheimer's disease (AD) one of the most devastating neurodegenerative disorders of our time. AD is associated with misfolding and formation of toxic aggregates of amyloid β (Aβ) peptide, subsequently producing β-rich amyloid deposits [1]. Evidence has emerged that amyloid fibrils may catalyse the process of corruptive misfolding through a positive-feed-back reaction loop [2]. However, detailed molecular mechanisms of this process remain elusive. In an effort to better understand molecular mechanisms of Aβ misfolding and fibrillization, we performed all-atom molecular dynamics (MD) simulations of interaction of Aβ<sub>1-42</sub> chains with fragments of β-rich amyloid fibrils in water. As the fibril models, we employed the solution NMR model 2NAO [3] and the cryo-EM model 5OQV [4]. Four disordered Aβ<sub>1-42</sub> chains were placed in random positions at a distance from the fibrils, and evolution of the entire systems was investigated. Multiple independent simulations were done for each system. For each fibril model, we observed attachment of Aβ<sub>1-42</sub> chains to the fibril. Importantly, both threading of Aβ<sub>1-42</sub> chains around the edge of the fibril (precursor of fibril elongation) and attachment of the chains to fibril's side surface (precursor of secondary nucleation) were detected with each of two fibril models. For the 5OQV model, threading around both the 'ridge' and 'groove' ends of the fibril were observed. Surprisingly, attachment of Aβ<sub>1-42</sub> chains to side surface of 5OQV fibrils occurred with the Aβ chains' preferential orientation along the fibril axis, rather than along cross-β strands. In one of simulations with the 2NAO fibril model, we observed detachment of an Aβ chain caused by its interaction with a different Aβ chain in solution (precursor of fibril dissociation). In contrast, the 5OQV fibrils have exhibited a remarkable structural stability in our simulations. In conclusion, the MD simulations have shed light into early stages of two major molecular mechanisms of misfolding and aggregation of Aβ<sub>1-42</sub>, fibril elongation and secondary misfolding, as well as captured a fibril dissociation event, revealing important molecular-level details behind these processes.

## References

[1] Eisenberg & Jucker, Cell 2012; 6:1188-1203.

[2] Cohen et al., PNAS 2013; 110:9758-9763.

[3] Walti et al., PNAS 2016; 113:E4976-E4984.

[4] Gremer et al., Science 2017; 358:116-119.

## 154. TSEs in European goats discriminated by Western blotting into five types based on five robust molecular parameters

L Pirisinuª, O Andreolettiᵇ, I Lantierᶜ, PL Acutisᵈ, C Acinᵉ, W Goldmannf, T Sklaviadisg, L Ekateriniadouh, C Fasti, P Papasavva – Stylianouj, S Simonk, J Spiropoulosl, U Agrimiª, JG Jacobsm, A Bossersm, LJM van Keulenm, M Mazza d, R Nonnoª and JPM Langeveldm

ªIstituto Superiore di Sanità, Department of Food Safety, Nutrition and Veterinary Public Health, Rome, Italy; ᵇUMR INRA ENVT 1225- IHAP, École Nationale Vétérinaire de Toulouse, Toulouse, France; ᶜINRA-Centre Val de Loire, Infectiologie et Santé Publique, Nouzilly, France; ᵈIstituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'Aosta, Torino, Italy; ᵉCentro de Encefalopatías y Enfermedades Transmisibles Emergentes, Facultad de Veterinaria, Universidad de Zaragoza, Zaragoza, Spain; ᶠThe Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, United Kingdom; ᵃLaboratory of Pharmacology, School of Health Sciences, Department of Pharmacy, Aristotle University of Thessaloniki, Thessaloniki, Greece; ᵇNational Agricultural Research Foundation, Veterinary Research Institute, Thessaloniki, Greece; ᵒInstitute of Novel and Emerging Infectious Diseases, Friedrich-Loeffler-Institute, Greifswald-Isle of Riems, Germany; ᵗVeterinary Services, Nicosia, Cyprus; ᵈService de Pharmacologie et Immunoanalyse (SPI), Laboratoire d'Etudes et de Recherches en Immunoanalyse, CEA, INRA, Université Paris-Saclay, Gif-sur-Yvette, France; ᵒAnimal and Plant Health Agency, New Haw, Addlestone, Surrey, United Kingdom; ᵒWageningen BioVeterinary Research, Lelystad, the Netherlands

CONTACT Laura Pirisinu laura.pirisinu@iss.it

## ABSTRACT

Contrasting the knowledge about prion diseases or TSEs in sheep, only a very limited number of strain typing studies are available in goats. Two cases deriving from the zoonotic bovine BSE epidemic were however detected in goats. During 2004–2012, over 70 TSE goat brain samples were collected from seven European countries and evaluated for TSE type/strain variation. A selection of these materials was chosen for in-depth analysis based on various criteria: tissue quality, genotype, broad geographical distribution, potential type variation. Of these, 37 cases were biochemically analysed (present study) and a subset of these subjected to bioassays in seven rodent models (see parallel study of Nonno et al.). Analyses of these isolates showed that in goats different PrPSc types exist, comparable to those found in sheep such as classical scrapie, CH1641-like scrapie and Nor98/atypical scrapie. However, classical scrapie isolates could be further differentiated in two molecular subtypes based on the PK susceptibility of the N-terminus and
<figure><img src="image_98.png" /></figure>

<sup>a</sup>Department of Electrical and Computer Engineering, University of Alberta, Edmonton, Canada; <sup>b</sup>National Institute for Nanotechnology NRC; Edmonton, Canada; <sup>c</sup>Department of Electrical and Computer Engineering, University of Alberta, Edmonton, Canada

CONTACT Lyudmyla Dorosh dorosh@ualberta.ca; Maria Stepanova ms1@ualberta.ca

## ABSTRACT

Prevalence in ageing population, difficulties in early diagnosis, and lack of efficient therapies make Alzheimer's disease (AD) one of the most devastating neurodegenerative disorders of our time. AD is associated with misfolding and formation of toxic aggregates of amyloid β (Aβ) peptide, subsequently producing β-rich amyloid deposits [1]. Evidence has emerged that amyloid fibrils may catalyse the process of corruptive misfolding through a positive-feed-back reaction loop [2]. However, detailed molecular mechanisms of this process remain elusive. In an effort to better understand molecular mechanisms of Aβ misfolding and fibrillization, we performed all-atom molecular dynamics (MD) simulations of interaction of Aβ<sub>1-42</sub> chains with fragments of β-rich amyloid fibrils in water. As the fibril models, we employed the solution NMR model 2NAO [3] and the cryo-EM model 5OQV [4]. Four disordered Aβ<sub>1-42</sub> chains were placed in random positions at a distance from the fibrils, and evolution of the entire systems was investigated. Multiple independent simulations were done for each system. For each fibril model, we observed attachment of Aβ<sub>1-42</sub> chains to the fibril. Importantly, both threading of Aβ<sub>1-42</sub> chains around the edge of the fibril (precursor of fibril elongation) and attachment of the chains to fibril's side surface (precursor of secondary nucleation) were detected with each of two fibril models. For the 5OQV model, threading around both the 'ridge' and 'groove' ends of the fibril were observed. Surprisingly, attachment of Aβ<sub>1-42</sub> chains to side surface of 5OQV fibrils occurred with the Aβ chains' preferential orientation along the fibril axis, rather than along cross-β strands. In one of simulations with the 2NAO fibril model, we observed detachment of an Aβ chain caused by its interaction with a different Aβ chain in solution (precursor of fibril dissociation). In contrast, the 5OQV fibrils have exhibited a remarkable structural stability in our simulations. In conclusion, the MD simulations have shed light into early stages of two major molecular mechanisms of misfolding and aggregation of Aβ<sub>1-42</sub>, fibril elongation and secondary misfolding, as well as captured a fibril dissociation event, revealing important molecular-level details behind these processes.

## References

[1] Eisenberg & Jucker, Cell 2012; 6:1188-1203.

[2] Cohen et al., PNAS 2013; 110:9758-9763.

[3] Walti et al., PNAS 2016; 113:E4976-E4984.

[4] Gremer et al., Science 2017; 358:116-119.

## 154. TSEs in European goats discriminated by Western blotting into five types based on five robust molecular parameters

L Pirisinuª, O Andreolettiᵇ, I Lantierᶜ, PL Acutisᵈ, C Acinᵉ, W Goldmannf, T Sklaviadisg, L Ekateriniadouh, C Fasti, P Papasavva – Stylianouj, S Simonk, J Spiropoulosl, U Agrimiª, JG Jacobsm, A Bossersm, LJM van Keulenm, M Mazza d, R Nonnoª and JPM Langeveldm

ªIstituto Superiore di Sanità, Department of Food Safety, Nutrition and Veterinary Public Health, Rome, Italy; ᵇUMR INRA ENVT 1225- IHAP, École Nationale Vétérinaire de Toulouse, Toulouse, France; ᶜINRA-Centre Val de Loire, Infectiologie et Santé Publique, Nouzilly, France; ᵈIstituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'Aosta, Torino, Italy; ᵉCentro de Encefalopatías y Enfermedades Transmisibles Emergentes, Facultad de Veterinaria, Universidad de Zaragoza, Zaragoza, Spain; ᶠThe Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, United Kingdom; ᵃLaboratory of Pharmacology, School of Health Sciences, Department of Pharmacy, Aristotle University of Thessaloniki, Thessaloniki, Greece; ᵇNational Agricultural Research Foundation, Veterinary Research Institute, Thessaloniki, Greece; ᵒInstitute of Novel and Emerging Infectious Diseases, Friedrich-Loeffler-Institute, Greifswald-Isle of Riems, Germany; ᵗVeterinary Services, Nicosia, Cyprus; ᵈService de Pharmacologie et Immunoanalyse (SPI), Laboratoire d'Etudes et de Recherches en Immunoanalyse, CEA, INRA, Université Paris-Saclay, Gif-sur-Yvette, France; ᵒAnimal and Plant Health Agency, New Haw, Addlestone, Surrey, United Kingdom; ᵒWageningen BioVeterinary Research, Lelystad, the Netherlands

CONTACT Laura Pirisinu laura.pirisinu@iss.it

## ABSTRACT

Contrasting the knowledge about prion diseases or TSEs in sheep, only a very limited number of strain typing studies are available in goats. Two cases deriving from the zoonotic bovine BSE epidemic were however detected in goats. During 2004–2012, over 70 TSE goat brain samples were collected from seven European countries and evaluated for TSE type/strain variation. A selection of these materials was chosen for in-depth analysis based on various criteria: tissue quality, genotype, broad geographical distribution, potential type variation. Of these, 37 cases were biochemically analysed (present study) and a subset of these subjected to bioassays in seven rodent models (see parallel study of Nonno et al.). Analyses of these isolates showed that in goats different PrPSc types exist, comparable to those found in sheep such as classical scrapie, CH1641-like scrapie and Nor98/atypical scrapie. However, classical scrapie isolates could be further differentiated in two molecular subtypes based on the PK susceptibility of the N-terminus and
<figure><img src="image_99.png" /></figure>

on the molecular weight of PrP<sup>res</sup>. Importantly, the two subtypes of classical scrapie showed different geographical distribution, as one subtype was only detected in goats deriving from Italy and France. These analyses also show, that none of the field samples exhibited BSE-like features and offer a comprehensive set of robust molecular PrP<sup>res</sup> properties to exclude suspicion for the presence of BSE. These are: molecular mass of the triplet bands, presence of N-terminus epitope, glycoprofile markers, a dual population marker and the structural stability of PrP-core.

This work was made possible by national support and
by the following European grants: Neuroprion (FP6-
FOOD-506,579), GoatBSE (FOOD-CT-2006-36,353) and
GOAT-TSE-FREE (EMIDA-ERA NET). We want to
memorize Jorg G Jacobs, who died in 2017. Jorg developed
and performed the Triplex-WB system as well as charac-
terized antibodies like 94B4, 12B2, 9A2, 6C2, SAF84 and
L42. He also was crucial in the distinction of C-, L- and H-
type BSE and in 2011 he showed elegantly the allotype
composition of prion material in heterozygous sheep by
the use of Endo-LysC.

155. Expert opinions on the potential role of
Indigenous peoples in wildlife management in
Alberta

Arlana Bennett

University of Alberta

CONTACT Arlana Bennett bennettc@ualberta.ca

ABSTRACT

**Background:** Natural resource management in many parts of the world are increasingly based around the recognition of Indigenous knowledge and the rights of resource users [1]. In Alberta, there has been a historical lack of consideration of the knowledge of First Nations and Métis peoples and decisions about the development, use, management and monitoring of natural resources [2–5]. As a result of these histories, Indigenous peoples and their knowledge have little influence within provincial wildlife management regimes in contemporary Alberta [6]. Wildlife diseases such as Chronic Wasting Disease (CWD) have added further challenges for wildlife managers in Alberta. CWD is a fatal form of transmissible spongiform encephalopathy (TSE) primarily found in cervids (e.g. deer, moose, elk, and caribou). CWD in Alberta as of the 2017/2018 hunting season, has continued to spread westward along the Red Deer/South

Saskatchewan/Bow watershed, the Battle watershed, and north along the Alberta/Saskatchewan border. Incidence of deer that have tested positive for CWD has increased markedly since the 2016/2017 hunting seasons, and several new Wildlife Management Units (WMU) will be added to the mandatory surveillance list for the 2018/19 hunting season [7]. First Nations and Métis communities in the Treaty 6 area – including Saddle Lake First Nation, Frog Lake First Nation, Kehewin First Nation, Fishing Lake Métis settlement, and the Elizabeth Métis settlement – are within the mandatory surveillance zone yet it remains unclear whether these communities are being actively engaged in the process of management.

**Methods:** This research sought to explore diverse expert perspectives on the role of Indigenous Knowledge in wildlife monitoring and management in relation to the issue of CWD; and better understand the key challenges and opportunities regarding wildlife management in Alberta. The methods used in this thesis include a modified qualitative expert elicitation, probabilistic sampling, and thematic analysis.

**Results:** The major thematic results experts discussed include: the lack of Indigenous compliance in cervid monitoring with varying reasons provided; the necessity of both scientists and Indigenous communities to engage in intercultural and technical capacity development; and the need for both scientists and Indigenous communities to form a functional and mutually beneficial working relationship. This research is a preliminary investigation into the social, cultural, and economic aspects of CWD management, and is intended to provide further insights towards this end with a focus on future areas of research.

References

[1] Berkes F. Sacred Ecology. 2018.

[2] Sandlos J. Hunters at the margin. 2007.

[3] Loo T. States of nature. 2006.

[4] Fumoleau R. As long as this land shall last. 2004.

[5] Calliou BL. Losing the game. 2000.

[6] Natcher, et al. Applied Anthropology. 2009;68:245–257.

[7] AEP (Alberta Environment and Parks). CWD Updates. 2018.

156. Screening and characterization of unusual
sCJD cases in a CWD endemic state in the USA

Yihui Liu<sup>a</sup>, Manuel Camacho<sup>a</sup>, Wenquan Zou<sup>a,b,c</sup>,
Qingzhong Kong<sup>a,b,c</sup>
<figure><img src="image_99.png" /></figure>

on the molecular weight of PrP<sup>res</sup>. Importantly, the two subtypes of classical scrapie showed different geographical distribution, as one subtype was only detected in goats deriving from Italy and France. These analyses also show, that none of the field samples exhibited BSE-like features and offer a comprehensive set of robust molecular PrP<sup>res</sup> properties to exclude suspicion for the presence of BSE. These are: molecular mass of the triplet bands, presence of N-terminus epitope, glycoprofile markers, a dual population marker and the structural stability of PrP-core.

This work was made possible by national support and
by the following European grants: Neuroprion (FP6-
FOOD-506,579), GoatBSE (FOOD-CT-2006-36,353) and
GOAT-TSE-FREE (EMIDA-ERA NET). We want to
memorize Jorg G Jacobs, who died in 2017. Jorg developed
and performed the Triplex-WB system as well as charac-
terized antibodies like 94B4, 12B2, 9A2, 6C2, SAF84 and
L42. He also was crucial in the distinction of C-, L- and H-
type BSE and in 2011 he showed elegantly the allotype
composition of prion material in heterozygous sheep by
the use of Endo-LysC.

155. Expert opinions on the potential role of
Indigenous peoples in wildlife management in
Alberta

Arlana Bennett

University of Alberta

CONTACT Arlana Bennett bennettc@ualberta.ca

ABSTRACT

**Background:** Natural resource management in many parts of the world are increasingly based around the recognition of Indigenous knowledge and the rights of resource users [1]. In Alberta, there has been a historical lack of consideration of the knowledge of First Nations and Métis peoples and decisions about the development, use, management and monitoring of natural resources [2–5]. As a result of these histories, Indigenous peoples and their knowledge have little influence within provincial wildlife management regimes in contemporary Alberta [6]. Wildlife diseases such as Chronic Wasting Disease (CWD) have added further challenges for wildlife managers in Alberta. CWD is a fatal form of transmissible spongiform encephalopathy (TSE) primarily found in cervids (e.g. deer, moose, elk, and caribou). CWD in Alberta as of the 2017/2018 hunting season, has continued to spread westward along the Red Deer/South

Saskatchewan/Bow watershed, the Battle watershed, and north along the Alberta/Saskatchewan border. Incidence of deer that have tested positive for CWD has increased markedly since the 2016/2017 hunting seasons, and several new Wildlife Management Units (WMU) will be added to the mandatory surveillance list for the 2018/19 hunting season [7]. First Nations and Métis communities in the Treaty 6 area – including Saddle Lake First Nation, Frog Lake First Nation, Kehewin First Nation, Fishing Lake Métis settlement, and the Elizabeth Métis settlement – are within the mandatory surveillance zone yet it remains unclear whether these communities are being actively engaged in the process of management.

**Methods:** This research sought to explore diverse expert perspectives on the role of Indigenous Knowledge in wildlife monitoring and management in relation to the issue of CWD; and better understand the key challenges and opportunities regarding wildlife management in Alberta. The methods used in this thesis include a modified qualitative expert elicitation, probabilistic sampling, and thematic analysis.

**Results:** The major thematic results experts discussed include: the lack of Indigenous compliance in cervid monitoring with varying reasons provided; the necessity of both scientists and Indigenous communities to engage in intercultural and technical capacity development; and the need for both scientists and Indigenous communities to form a functional and mutually beneficial working relationship. This research is a preliminary investigation into the social, cultural, and economic aspects of CWD management, and is intended to provide further insights towards this end with a focus on future areas of research.

References

[1] Berkes F. Sacred Ecology. 2018.

[2] Sandlos J. Hunters at the margin. 2007.

[3] Loo T. States of nature. 2006.

[4] Fumoleau R. As long as this land shall last. 2004.

[5] Calliou BL. Losing the game. 2000.

[6] Natcher, et al. Applied Anthropology. 2009;68:245–257.

[7] AEP (Alberta Environment and Parks). CWD Updates. 2018.

156. Screening and characterization of unusual
sCJD cases in a CWD endemic state in the USA

Yihui Liu<sup>a</sup>, Manuel Camacho<sup>a</sup>, Wenquan Zou<sup>a,b,c</sup>,
Qingzhong Kong<sup>a,b,c</sup>
<figure><img src="image_100.png" /></figure>

<sup>a</sup>Department of Pathology, Case Western Reserve University (CWRU), Cleveland, USA; <sup>b</sup>Department of Neurology, CWRU, Cleveland, OH, USA; <sup>c</sup>National Center for Regenerative Medicine, CWRU, Cleveland, USA

CONTACT Qingzhong Kong qxk2@case.edu

## ABSTRACT

**Background:** Chronic wasting disease (CWD) has spread to 26 states in the USA and three provinces in Canada, and it has been detected recently in Norway and Finland. Potential CWD zoonosis is a serious public health concern. It is unclear whether CWD transmission to humans has already occurred. We aim to start to address this question by examining all available sCJD cases from a CWD endemic state in the USA.

**Methods:** Frozen brain tissues from all available sCJD cases archived in the National Prion Disease Pathology Surveillance Center from a US state that has been significantly impacted by CWD were sampled at five brain regions. These brain samples were subjected to detailed biochemical analysis to look for unusual patterns, characteristics, and/or distribution of $PrP^{Sc}$ in comparison with sCJD samples from states that have not detected CWD. Unusual cases are further scrutinized for their clinical presentations, histopathological features, and history of cervid hunting and venison consumption.

**Results and Conclusions:** We have found some unusual sCJD cases in this CWD endemic state. We will report our preliminary findings on their features. Currently there is no convincing evidence to support a direct link to CWD for any of these unusual sCJD cases.

**Disclaimer:** The findings and conclusions in this report are those of the authors and do not necessarily represent the position of the National Prion Disease Pathology Surveillance Center.

## 157. Utility of RT-QuIC in predicting case status of potential Creutzfeldt-Jakob Disease (CJD) cases

Natalie S. Marzec<sup>a</sup>, Marie-Ange Smith<sup>b</sup>, Jennifer A. House<sup>a</sup>

<sup>a</sup>Colorado Department of Public Health and Environment, Denver, CO, USA; <sup>b</sup>Colorado School of Public Health, Aurora, CO, USA

CONTACT Natalie S. Marzec natalie.marzec@state.co.us

## ABSTRACT

**Background:** Ante-mortem diagnosis of human prion disease is limited by the high false positivity rate of the testing methods available for cerebrospinal fluid (CSF). This has resulted in a lack of efficiency when

investigating potential prion disease cases for surveillance purposes. In 2018, the real time quake-inducing conversion (RT-QuIC) test was added to the CDC diagnostic criteria for Creutzfeldt-Jakob Disease (CJD), the most common prion disease reported in humans. RT-QuIC appears to be a better predictor of Definite or Probable CJD case status than the older Tau and 14-3-3 protein tests. The purpose of this project was to evaluate the utility of RT-QuIC to predict case status of potential CJD cases.

**Methods:** Potential cases of CJD reported in Colorado between 1 January 2015, and 31 December 2017 (prior to the change in the CDC diagnostic criteria), were reviewed for final case status, testing methods, and ante-mortem test results. RT-QuIC, Tau, and 14-3-3 test results were compared to final case status to determine the positive predictive value (PPV), negative predictive value (NPV), sensitivity, and specificity of each test. Cases that met either Definite or Probable case definitions were considered to be true cases.

**Results:** Sixty-six potential cases of CJD reported in the specified time period were investigated and assigned a case status of Definite, Probable, Possible, or did not meet case definition. Of these cases, 27 (41%) were assigned a Definite or Probable case status. RT-QuIC was found to have a PPV of 92.8%, a NPV of 100%, a sensitivity of 100% and a specificity of 95.8%. Tau protein had a PPV of 48.6%, a NPV of 93.8%, a sensitivity of 94.7%, and a specificity of 55.9%. 14-3-3 protein had a PPV of 32.4%, a NPV of 83.3%, a sensitivity of 92.3%, and a specificity of 16.7%.

**Conclusions:** RT-QuIC testing is a more useful ante-mortem predictor of Definite or Probable CJD case status than either Tau or 14-3-3 protein testing. Public health investigators can place more weight on RT-QuIC testing than other ante-mortem CJD disease testing when triaging cases for investigation. To our knowledge, this is the first time RT-QuIC testing has been studied in relation to predicting public health surveillance case status.

**KEYWORDS:** Creutzfeldt-Jakob disease; RT-QuIC; public health; surveillance

## 158. Familial Creutzfeldt-Jakob disease with D178N and Met129Val

Nurit Omer<sup>a</sup>, Esther Kahana<sup>b</sup>, Sharon Simhoni<sup>c</sup>, Anat Bar-Shira<sup>c</sup>, Tova Naiman<sup>c</sup>, Avi Orr-Urtreger<sup>c</sup>, Nir Giladi<sup>d</sup> and Noa Bregman<sup>a</sup>

<sup>a</sup>CJD clinic, Neurological institute, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel; <sup>b</sup>Department of Neurology, Barzilai Medical Center, Ashkelon, Israel; <sup>c</sup>The Genetic Institute, Tel-Aviv Sourasky Medical Center, Tel-Aviv, Israel; <sup>d</sup>Neurological institute, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel
<figure><img src="image_100.png" /></figure>

<sup>a</sup>Department of Pathology, Case Western Reserve University (CWRU), Cleveland, USA; <sup>b</sup>Department of Neurology, CWRU, Cleveland, OH, USA; <sup>c</sup>National Center for Regenerative Medicine, CWRU, Cleveland, USA

CONTACT Qingzhong Kong qxk2@case.edu

## ABSTRACT

**Background:** Chronic wasting disease (CWD) has spread to 26 states in the USA and three provinces in Canada, and it has been detected recently in Norway and Finland. Potential CWD zoonosis is a serious public health concern. It is unclear whether CWD transmission to humans has already occurred. We aim to start to address this question by examining all available sCJD cases from a CWD endemic state in the USA.

**Methods:** Frozen brain tissues from all available sCJD cases archived in the National Prion Disease Pathology Surveillance Center from a US state that has been significantly impacted by CWD were sampled at five brain regions. These brain samples were subjected to detailed biochemical analysis to look for unusual patterns, characteristics, and/or distribution of $PrP^{Sc}$ in comparison with sCJD samples from states that have not detected CWD. Unusual cases are further scrutinized for their clinical presentations, histopathological features, and history of cervid hunting and venison consumption.

**Results and Conclusions:** We have found some unusual sCJD cases in this CWD endemic state. We will report our preliminary findings on their features. Currently there is no convincing evidence to support a direct link to CWD for any of these unusual sCJD cases.

**Disclaimer:** The findings and conclusions in this report are those of the authors and do not necessarily represent the position of the National Prion Disease Pathology Surveillance Center.

## 157. Utility of RT-QuIC in predicting case status of potential Creutzfeldt-Jakob Disease (CJD) cases

Natalie S. Marzec<sup>a</sup>, Marie-Ange Smith<sup>b</sup>, Jennifer A. House<sup>a</sup>

<sup>a</sup>Colorado Department of Public Health and Environment, Denver, CO, USA; <sup>b</sup>Colorado School of Public Health, Aurora, CO, USA

CONTACT Natalie S. Marzec natalie.marzec@state.co.us

## ABSTRACT

**Background:** Ante-mortem diagnosis of human prion disease is limited by the high false positivity rate of the testing methods available for cerebrospinal fluid (CSF). This has resulted in a lack of efficiency when

investigating potential prion disease cases for surveillance purposes. In 2018, the real time quake-inducing conversion (RT-QuIC) test was added to the CDC diagnostic criteria for Creutzfeldt-Jakob Disease (CJD), the most common prion disease reported in humans. RT-QuIC appears to be a better predictor of Definite or Probable CJD case status than the older Tau and 14-3-3 protein tests. The purpose of this project was to evaluate the utility of RT-QuIC to predict case status of potential CJD cases.

**Methods:** Potential cases of CJD reported in Colorado between 1 January 2015, and 31 December 2017 (prior to the change in the CDC diagnostic criteria), were reviewed for final case status, testing methods, and ante-mortem test results. RT-QuIC, Tau, and 14-3-3 test results were compared to final case status to determine the positive predictive value (PPV), negative predictive value (NPV), sensitivity, and specificity of each test. Cases that met either Definite or Probable case definitions were considered to be true cases.

**Results:** Sixty-six potential cases of CJD reported in the specified time period were investigated and assigned a case status of Definite, Probable, Possible, or did not meet case definition. Of these cases, 27 (41%) were assigned a Definite or Probable case status. RT-QuIC was found to have a PPV of 92.8%, a NPV of 100%, a sensitivity of 100% and a specificity of 95.8%. Tau protein had a PPV of 48.6%, a NPV of 93.8%, a sensitivity of 94.7%, and a specificity of 55.9%. 14-3-3 protein had a PPV of 32.4%, a NPV of 83.3%, a sensitivity of 92.3%, and a specificity of 16.7%.

**Conclusions:** RT-QuIC testing is a more useful ante-mortem predictor of Definite or Probable CJD case status than either Tau or 14-3-3 protein testing. Public health investigators can place more weight on RT-QuIC testing than other ante-mortem CJD disease testing when triaging cases for investigation. To our knowledge, this is the first time RT-QuIC testing has been studied in relation to predicting public health surveillance case status.

**KEYWORDS:** Creutzfeldt-Jakob disease; RT-QuIC; public health; surveillance

## 158. Familial Creutzfeldt-Jakob disease with D178N and Met129Val

Nurit Omer<sup>a</sup>, Esther Kahana<sup>b</sup>, Sharon Simhoni<sup>c</sup>, Anat Bar-Shira<sup>c</sup>, Tova Naiman<sup>c</sup>, Avi Orr-Urtreger<sup>c</sup>, Nir Giladi<sup>d</sup> and Noa Bregman<sup>a</sup>

<sup>a</sup>CJD clinic, Neurological institute, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel; <sup>b</sup>Department of Neurology, Barzilai Medical Center, Ashkelon, Israel; <sup>c</sup>The Genetic Institute, Tel-Aviv Sourasky Medical Center, Tel-Aviv, Israel; <sup>d</sup>Neurological institute, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel
<figure><img src="image_101.png" /></figure>

CONTACT Nurit Omer nurito@tlvmc.gov.il

ABSTRACT

Fatal familial insomnia (FFI) and Creutzfeldt-Jakob disease (CJD) are two phenotypes that share a common point mutation at codon 178 of the prion protein gene (PRNP), with the substitution of aspartic acid to asparagine (D178N). The phenotype depends on the polymorphism at codon 129, when methionine (129M) is associated with FFI, and Valine (129V) with CJD. We here report on a Jewish family from Iraqi origin with D178N-129V genotype presenting with overt insomnia, cerebellar ataxia, and an atypical duration of disease. Patient 1, a 56-year-old woman without family history of neurological illness, presented in 1996 with rapidly progressive dementia and gait ataxia. MRI showed frontal cortical but not basal ganglia or thalamic involvement. CSF was positive for protein 14-3-3, and genetic testing for E200K mutation was negative. Brain biopsy showed typical spongiform changes, and she was diagnosed with sporadic CJD. She passed away 7 years after symptoms onset. Her son (patient 2) presented on 2012 with rapidly progressive dementia at the age of 41 and was diagnosed with CJD. He died a few months later. Patient 3, her daughter, presented on 2018 at the age of 50, with cerebellar ataxia and mild cognitive impairment. She described severe insomnia for more than a year before arrival. Brain MRI showed overt restriction in diffusion across the frontal, parietal and temporal cortices, with marked involvement of basal ganglia and thalami. A genetic analysis of the PRNP gene revealed a heterozygous D178N mutation (sequence variant: c.532G>A) in combination with the polymorphism Met129Val (sequence variant: c.385A>G) on both alleles (homozygous). Patient 4, her third daughter, 45 years old, is currently (2019) undergoing medical evaluation due to reported progressive cognitive decline for 6 months. The phenotype of patient 1 was clearly different from that of FFI and more suggestive of CJD, although the long duration of the disease is highly unusual for CJD. Severe insomnia was reported in the 'prodromal' phase in patient 3, with marked thalami involvement in MRI, but with a genetic signature that is consistent with CJD. Our findings support the notion that there is a phenotypic variability in the D178N genotype within the same family, and emphasize the heterogeneity of inherited prion disease and the possibility that genetic and other modifiers exist.

159. Therapeutic and clinical strategy to prevent or delay onset of genetic prion disease using prion protein-lowering antisense oligonucleotides

Eric Vallabh Minikel<sup>a,b,c</sup>, Hien Tran Zhao<sup>d</sup>, Deb Cabin<sup>e</sup>, Stuart L. Schreiber<sup>a,f</sup>, Jeffrey B. Carroll<sup>e</sup>, Holly

Kordasiewicz<sup>d,g</sup>, Steven E. Arnold<sup>b,h</sup>, Byron Caughey<sup>i</sup> and Sonia M. Vallabh<sup>a,b,c</sup>

<sup>a</sup>Broad Institute of MIT and Harvard, Cambridge, MA, USA; <sup>b</sup>Harvard Medical School, Boston, MA, USA; <sup>c</sup>Prion Alliance, Cambridge, MA, USA; <sup>d</sup>Ionis Pharmaceuticals Inc, Carlsbad, CA, USA; <sup>e</sup>McLaughlin Research Institute, Great Falls, MT, USA; <sup>f</sup>Harvard University, Department of Chemistry and Chemical Biology, Cambridge, MA, USA; <sup>g</sup>Western Washington University, Bellingham, WA, USA; <sup>h</sup>Massachusetts General Hospital, Department of Neurology, Boston, MA, USA; <sup>i</sup>Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT, USA

CONTACT Sonia M. Vallabh svallabh@broadinstitute.org

ABSTRACT

**Background:** Genetic proofs of concept suggest that reduction of prion protein (PrP) in the brain should be an effective and well tolerated therapeutic strategy for prion disease. We are therefore investigating PrP-lowering antisense oligonucleotides (ASOs) as a potential prion disease therapeutic. Based on initial observations of ASO efficacy in prion-infected mice (see complementary abstract by Dr Byron Caughey) here we assess parameters of ASO efficacy including dose responsiveness, strain specificity, and impact of treatment timepoint. As early treatment conveys greater benefit, we also describe a framework for testing PrP-lowering therapeutics in pre-symptomatic prion disease mutation carriers, including biomarker development, establishment of a carrier cohort and regulatory engagement.

**Materials and methods:** All mice received bolus doses of active ASOs or saline by stereotactic intracerebroventricular injection, then were monitored for symptoms with a primary outcome of terminal prion disease. ASO doses, prion strains and treatment initiation timepoint varied across experiments. To evaluate CSF PrP as a biomarker for PrP-lowering therapeutics, *N* = 217 human CSF samples spanning a range of diagnoses and prion disease subtypes were subjected to PrP quantification by ELISA.

**Results:** ASO efficacy is dose responsive, universal across all prion strains tested, and more efficacious if administered early. In an ongoing experiment, PrP-lowering ASOs were administered every 90 days beginning at -14 dpi; all saline-treated animals have reached prion endpoint (mean 143 dpi) while all but one ASO-treated animal is living at 316 dpi, corresponding to a ≥ 2.2x extension of survival. CSF PrP is detectable in all human CSF samples tested, reliably quantifiable by ELISA subject to proper handling, appears CNS-derived, and exhibits excellent short-term within-subject test-retest reliability (mean CV = 8.5%) in genetic prion disease mutation carriers, suggesting that ASO-mediated PrP lowering should be readily detectable in the context of a trial. In a recent Critical Path Innovation
<figure><img src="image_101.png" /></figure>

CONTACT Nurit Omer nurito@tlvmc.gov.il

ABSTRACT

Fatal familial insomnia (FFI) and Creutzfeldt-Jakob disease (CJD) are two phenotypes that share a common point mutation at codon 178 of the prion protein gene (PRNP), with the substitution of aspartic acid to asparagine (D178N). The phenotype depends on the polymorphism at codon 129, when methionine (129M) is associated with FFI, and Valine (129V) with CJD. We here report on a Jewish family from Iraqi origin with D178N-129V genotype presenting with overt insomnia, cerebellar ataxia, and an atypical duration of disease. Patient 1, a 56-year-old woman without family history of neurological illness, presented in 1996 with rapidly progressive dementia and gait ataxia. MRI showed frontal cortical but not basal ganglia or thalamic involvement. CSF was positive for protein 14-3-3, and genetic testing for E200K mutation was negative. Brain biopsy showed typical spongiform changes, and she was diagnosed with sporadic CJD. She passed away 7 years after symptoms onset. Her son (patient 2) presented on 2012 with rapidly progressive dementia at the age of 41 and was diagnosed with CJD. He died a few months later. Patient 3, her daughter, presented on 2018 at the age of 50, with cerebellar ataxia and mild cognitive impairment. She described severe insomnia for more than a year before arrival. Brain MRI showed overt restriction in diffusion across the frontal, parietal and temporal cortices, with marked involvement of basal ganglia and thalami. A genetic analysis of the PRNP gene revealed a heterozygous D178N mutation (sequence variant: c.532G>A) in combination with the polymorphism Met129Val (sequence variant: c.385A>G) on both alleles (homozygous). Patient 4, her third daughter, 45 years old, is currently (2019) undergoing medical evaluation due to reported progressive cognitive decline for 6 months. The phenotype of patient 1 was clearly different from that of FFI and more suggestive of CJD, although the long duration of the disease is highly unusual for CJD. Severe insomnia was reported in the 'prodromal' phase in patient 3, with marked thalami involvement in MRI, but with a genetic signature that is consistent with CJD. Our findings support the notion that there is a phenotypic variability in the D178N genotype within the same family, and emphasize the heterogeneity of inherited prion disease and the possibility that genetic and other modifiers exist.

159. Therapeutic and clinical strategy to prevent or delay onset of genetic prion disease using prion protein-lowering antisense oligonucleotides

Eric Vallabh Minikel<sup>a,b,c</sup>, Hien Tran Zhao<sup>d</sup>, Deb Cabin<sup>e</sup>, Stuart L. Schreiber<sup>a,f</sup>, Jeffrey B. Carroll<sup>e</sup>, Holly

Kordasiewicz<sup>d,g</sup>, Steven E. Arnold<sup>b,h</sup>, Byron Caughey<sup>i</sup> and Sonia M. Vallabh<sup>a,b,c</sup>

<sup>a</sup>Broad Institute of MIT and Harvard, Cambridge, MA, USA; <sup>b</sup>Harvard Medical School, Boston, MA, USA; <sup>c</sup>Prion Alliance, Cambridge, MA, USA; <sup>d</sup>Ionis Pharmaceuticals Inc, Carlsbad, CA, USA; <sup>e</sup>McLaughlin Research Institute, Great Falls, MT, USA; <sup>f</sup>Harvard University, Department of Chemistry and Chemical Biology, Cambridge, MA, USA; <sup>g</sup>Western Washington University, Bellingham, WA, USA; <sup>h</sup>Massachusetts General Hospital, Department of Neurology, Boston, MA, USA; <sup>i</sup>Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT, USA

CONTACT Sonia M. Vallabh svallabh@broadinstitute.org

ABSTRACT

**Background:** Genetic proofs of concept suggest that reduction of prion protein (PrP) in the brain should be an effective and well tolerated therapeutic strategy for prion disease. We are therefore investigating PrP-lowering antisense oligonucleotides (ASOs) as a potential prion disease therapeutic. Based on initial observations of ASO efficacy in prion-infected mice (see complementary abstract by Dr Byron Caughey) here we assess parameters of ASO efficacy including dose responsiveness, strain specificity, and impact of treatment timepoint. As early treatment conveys greater benefit, we also describe a framework for testing PrP-lowering therapeutics in pre-symptomatic prion disease mutation carriers, including biomarker development, establishment of a carrier cohort and regulatory engagement.

**Materials and methods:** All mice received bolus doses of active ASOs or saline by stereotactic intracerebroventricular injection, then were monitored for symptoms with a primary outcome of terminal prion disease. ASO doses, prion strains and treatment initiation timepoint varied across experiments. To evaluate CSF PrP as a biomarker for PrP-lowering therapeutics, *N* = 217 human CSF samples spanning a range of diagnoses and prion disease subtypes were subjected to PrP quantification by ELISA.

**Results:** ASO efficacy is dose responsive, universal across all prion strains tested, and more efficacious if administered early. In an ongoing experiment, PrP-lowering ASOs were administered every 90 days beginning at -14 dpi; all saline-treated animals have reached prion endpoint (mean 143 dpi) while all but one ASO-treated animal is living at 316 dpi, corresponding to a ≥ 2.2x extension of survival. CSF PrP is detectable in all human CSF samples tested, reliably quantifiable by ELISA subject to proper handling, appears CNS-derived, and exhibits excellent short-term within-subject test-retest reliability (mean CV = 8.5%) in genetic prion disease mutation carriers, suggesting that ASO-mediated PrP lowering should be readily detectable in the context of a trial. In a recent Critical Path Innovation
<figure><img src="image_102.png" /></figure>

Meeting, we discussed with FDA scientists a path to pre-symptomatic trials in genetic prion disease mutation carriers based on CSF PrP lowering as surrogate endpoint for provisional approval of an ASO.

**Conclusions:** These data support advancement of PrP-lowering ASOs to the clinic, and support the feasibility of primary prevention trials in healthy genetic prion disease mutation carriers. We are working closely with our pharmaceutical partner, Ionis Pharmaceuticals, as well as with FDA to advance ASOs to the clinic within the next few years. Genetic prion disease is poised to become a model for primary prevention in neurodegeneration.

## 160. Rapid bioassay of mammalian prions in Drosophila

Alana M. Thackray and Raymond Bujdoso

Department of Veterinary Medicine, Cambridge University,
Cambridge, UK

CONTACT Raymond Bujdoso rb202@cam.ac.uk

### ABSTRACT

Mammalian prions cause fatal neurodegenerative diseases such as Creutzfeldt-Jakob disease (CJD) in humans, Bovine Spongiform Encephalopathy (BSE) in cattle, chronic wasting disease (CWD) in cervids and scrapie disease of sheep. These conditions are transmissible between individuals of the same or different species and as a consequence, animal prion diseases pose a realistic threat to human health through their zoonotic potential. Prions lack a conventional nucleic acid-based genome. The 'protein only' concept predicts that infectious prion particles consist of $PrP^{Sc}$ in the form of aggregates of misfolded conformers of the normal host protein $PrP^{C}$, although the exact molecular nature of this transmissible entity is undefined. As a consequence, the only reliable method to detect mammalian prion infectivity is by bioassay in an appropriate experimental host. It is important to be able to detect prion infectivity in tissues and fluids from individuals affected by prion disease in order to protect human and animal health. In addition, it is important to address the zoonotic potential of animal prion infectivity in order to ensure the safety of the human food chain. The detection of prion infectivity by bioassay in mammalian species, most commonly mice, is time consuming and expensive, and consequently, low numbers of samples are routinely assessed. We have previously demonstrated *Drosophila* can bioassay mammalian prions (Thackray, et al. Brain 2018; 141:2700-2710). Our studies have shown that ovine PrP transgenic *Drosophila* develop a transmissible neurotoxic phenotype, associated with Proteinase-K resistant $PrP^{Sc}$,

after exposure to exogenous ovine prions. We have recently generated *Drosophila* transgenic for human, bovine or cer vid PrP and have demonstrated that these flies can be used successfully to bioassay variant CJD, BSE or CWD samples, respectively. In addition, we have shown that human PrP transgenic *Drosophila* can be used to model the zoonotic potential of animal prions. Collectively, our novel data show that *Drosophila* can detect human and animal prion infectivity in a rapid and efficient manner, and can con tribute to an understanding of the zoonotic potential of animal prion disease.

## 161. Rodent models allow BSE discrimination of goat prions and reveal geographical differences in the biological properties of scrapie

R. Nonno<sup>a</sup>, A. Marin-Moreno<sup>b</sup>, J.C. Espinosa<sup>b</sup>, C. Fast<sup>c</sup>, L. Van Keulen<sup>d</sup>, J. Spiropoulos<sup>e</sup>, I. Lantier<sup>f</sup>, O. Andreoletti<sup>g</sup>, L. Pirisinu<sup>a</sup>, M.A. Di Bari<sup>a</sup>, P. Aguilar-Calvo<sup>b</sup>, T. Sklaviadis<sup>h</sup>, P. Papasavva-Stylianou<sup>i</sup>, P.L. Acutis<sup>j</sup>, C. Acin<sup>k</sup>, A. Bossers<sup>d</sup>, J.G. Jacobs<sup>d</sup>, G. Vaccari<sup>a</sup>, C. D'Agostino<sup>a</sup>, B. Chiappini<sup>a</sup>, F. Lantier<sup>f</sup>, M. Groschup<sup>c</sup>, U. Agrimi<sup>a</sup>, J.M. Torres<sup>b</sup> and J.P.M. Langeveld<sup>d</sup>

<sup>a</sup>Istituto Superiore di Sanità, Department of Food Safety, Nutrition and Veterinary Public Health, Rome, Italy;
<sup>b</sup>Centro de Investigación en Sanidad Animal, CISA-INIA, Madrid, Spain;
<sup>c</sup>Institute of Novel and Emerging Infectious Diseases, Friedrich-Loeffler-Institute, Greifswald-Isle of Riems, Germany;
<sup>d</sup>Wageningen BioVeterinary Research, Lelystad, the Netherlands;
<sup>e</sup>Animal and Plant Health Agency, New Haw, Addlestone, Surrey, United Kingdom;
<sup>f</sup>INRA-Centre Val de Loire, Infectiologie et Santé Publique, Nouzilly, France;
<sup>g</sup>UMR INRA ENVT 1225- IHAP, École Nationale Vétérinaire de Toulouse, Toulouse, France;
<sup>h</sup>Laboratory of Pharmacology, School of Health Sciences, Department of Pharmacy, Aristotle University of Thessaloniki, Thessaloniki, Greece;
<sup>i</sup>Veterinary Services, Nicosia, Cyprus;
<sup>j</sup>Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'Aosta, Torino, Italy;
<sup>k</sup>Centro de Encefalopatías y Enfermedades Transmisibles Emergentes, Facultad de Veterinaria, Universidad de Zaragoza, Zaragoza, Spain

CONTACT Romolo Nonno romolo.nonno@iss.it

### ABSTRACT

Classical scrapie (CS) is a contagious TSE of small ruminants known to circulate in Europe for centuries. More than one TSE strain is responsible for CS, still our ability to identify scrapie strains remains limited, as it has been difficult so far to reconcile data obtained in different rodent models. After the discovery of BSE in two goats, a European-broad effort was undertaken in
<figure><img src="image_102.png" /></figure>

Meeting, we discussed with FDA scientists a path to pre-symptomatic trials in genetic prion disease mutation carriers based on CSF PrP lowering as surrogate endpoint for provisional approval of an ASO.

**Conclusions:** These data support advancement of PrP-lowering ASOs to the clinic, and support the feasibility of primary prevention trials in healthy genetic prion disease mutation carriers. We are working closely with our pharmaceutical partner, Ionis Pharmaceuticals, as well as with FDA to advance ASOs to the clinic within the next few years. Genetic prion disease is poised to become a model for primary prevention in neurodegeneration.

## 160. Rapid bioassay of mammalian prions in Drosophila

Alana M. Thackray and Raymond Bujdoso

Department of Veterinary Medicine, Cambridge University,
Cambridge, UK

CONTACT Raymond Bujdoso rb202@cam.ac.uk

### ABSTRACT

Mammalian prions cause fatal neurodegenerative diseases such as Creutzfeldt-Jakob disease (CJD) in humans, Bovine Spongiform Encephalopathy (BSE) in cattle, chronic wasting disease (CWD) in cervids and scrapie disease of sheep. These conditions are transmissible between individuals of the same or different species and as a consequence, animal prion diseases pose a realistic threat to human health through their zoonotic potential. Prions lack a conventional nucleic acid-based genome. The 'protein only' concept predicts that infectious prion particles consist of $PrP^{Sc}$ in the form of aggregates of misfolded conformers of the normal host protein $PrP^{C}$, although the exact molecular nature of this transmissible entity is undefined. As a consequence, the only reliable method to detect mammalian prion infectivity is by bioassay in an appropriate experimental host. It is important to be able to detect prion infectivity in tissues and fluids from individuals affected by prion disease in order to protect human and animal health. In addition, it is important to address the zoonotic potential of animal prion infectivity in order to ensure the safety of the human food chain. The detection of prion infectivity by bioassay in mammalian species, most commonly mice, is time consuming and expensive, and consequently, low numbers of samples are routinely assessed. We have previously demonstrated *Drosophila* can bioassay mammalian prions (Thackray, et al. Brain 2018; 141:2700-2710). Our studies have shown that ovine PrP transgenic *Drosophila* develop a transmissible neurotoxic phenotype, associated with Proteinase-K resistant $PrP^{Sc}$,

after exposure to exogenous ovine prions. We have recently generated *Drosophila* transgenic for human, bovine or cer vid PrP and have demonstrated that these flies can be used successfully to bioassay variant CJD, BSE or CWD samples, respectively. In addition, we have shown that human PrP transgenic *Drosophila* can be used to model the zoonotic potential of animal prions. Collectively, our novel data show that *Drosophila* can detect human and animal prion infectivity in a rapid and efficient manner, and can con tribute to an understanding of the zoonotic potential of animal prion disease.

## 161. Rodent models allow BSE discrimination of goat prions and reveal geographical differences in the biological properties of scrapie

R. Nonno<sup>a</sup>, A. Marin-Moreno<sup>b</sup>, J.C. Espinosa<sup>b</sup>, C. Fast<sup>c</sup>, L. Van Keulen<sup>d</sup>, J. Spiropoulos<sup>e</sup>, I. Lantier<sup>f</sup>, O. Andreoletti<sup>g</sup>, L. Pirisinu<sup>a</sup>, M.A. Di Bari<sup>a</sup>, P. Aguilar-Calvo<sup>b</sup>, T. Sklaviadis<sup>h</sup>, P. Papasavva-Stylianou<sup>i</sup>, P.L. Acutis<sup>j</sup>, C. Acin<sup>k</sup>, A. Bossers<sup>d</sup>, J.G. Jacobs<sup>d</sup>, G. Vaccari<sup>a</sup>, C. D'Agostino<sup>a</sup>, B. Chiappini<sup>a</sup>, F. Lantier<sup>f</sup>, M. Groschup<sup>c</sup>, U. Agrimi<sup>a</sup>, J.M. Torres<sup>b</sup> and J.P.M. Langeveld<sup>d</sup>

<sup>a</sup>Istituto Superiore di Sanità, Department of Food Safety, Nutrition and Veterinary Public Health, Rome, Italy;
<sup>b</sup>Centro de Investigación en Sanidad Animal, CISA-INIA, Madrid, Spain;
<sup>c</sup>Institute of Novel and Emerging Infectious Diseases, Friedrich-Loeffler-Institute, Greifswald-Isle of Riems, Germany;
<sup>d</sup>Wageningen BioVeterinary Research, Lelystad, the Netherlands;
<sup>e</sup>Animal and Plant Health Agency, New Haw, Addlestone, Surrey, United Kingdom;
<sup>f</sup>INRA-Centre Val de Loire, Infectiologie et Santé Publique, Nouzilly, France;
<sup>g</sup>UMR INRA ENVT 1225- IHAP, École Nationale Vétérinaire de Toulouse, Toulouse, France;
<sup>h</sup>Laboratory of Pharmacology, School of Health Sciences, Department of Pharmacy, Aristotle University of Thessaloniki, Thessaloniki, Greece;
<sup>i</sup>Veterinary Services, Nicosia, Cyprus;
<sup>j</sup>Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'Aosta, Torino, Italy;
<sup>k</sup>Centro de Encefalopatías y Enfermedades Transmisibles Emergentes, Facultad de Veterinaria, Universidad de Zaragoza, Zaragoza, Spain

CONTACT Romolo Nonno romolo.nonno@iss.it

### ABSTRACT

Classical scrapie (CS) is a contagious TSE of small ruminants known to circulate in Europe for centuries. More than one TSE strain is responsible for CS, still our ability to identify scrapie strains remains limited, as it has been difficult so far to reconcile data obtained in different rodent models. After the discovery of BSE in two goats, a European-broad effort was undertaken in
<figure><img src="image_103.png" /></figure>

order to understand the diversity of goat TSE strains and to discriminate BSE from them. Here, we studied goat TSEs by their relative transmission efficiency in different animal models and by the $PrP^{Sc}$ type(s) propagated in rodents. Thus, the biological properties of TSE isolates were inferred by their specific interaction with different recipient PrP species rather than by serial passage in a single model. Goat TSE isolates from seven countries and experimental goat-BSE were inoculated in seven models: tg-mice overexpressing sheep/goat ARQ, sheep VRQ, cattle or mouse PrPs; RIII mice and bank voles. The attack rate and the survival time were combined to obtain the 'transmission efficiency', a parameter used to build the 'transmission profiles' of individual isolates across all models. Goat-BSE and Nor98 showed distinctive transmission profiles, different among them and from all other isolates. Goat-BSE was the only isolate inducing 19kDa $PrP^{Sc}$ in all rodent models, while Nor98 was associated with 8kDa $PrP^{Sc}$. CS isolates showed a strong variability of transmission profiles, which was in part explained by their geographical distribution, and could be grouped into four distinct categories. CS isolates in category 1 induced the propagation solely of 21kDa $PrP^{Sc}$, while isolates from CS categories 2-to-4 also induced, to a different extent depending on the model, a 19kDa $PrP^{Sc}$ type distinct from BSE. Further investigation of CNS and LRS tissues from a goat herd showed that this phenomenon reflects the selective amplification in rodents of at least two co-existing 'sub-strain components', 21kDa and 19kDa, whose relative amount varies with different goat tissues/isolates. Our findings show that BSE can be discriminated from a wide range of biologically and geographically diverse goat TSEs. The geographical variation observed in CS suggests the existence of distinct CS strains, as also supported by the parallel studies by Pirisinu et al. and Marin-Moreno et al. Yet, CS isolates in categories 2-to-4 were mixtures of discrete sub-strains, where it could be envisaged that competition among sub-strains play a role in defining the biological properties of individual isolates and their evolution.

## 162. 'Multi-species anti-prion oral vaccine for familial and transmissible spongiform encephalopathies'

Fernando Goñiª, Analia Eliseiᵇ, Lucia Yimᶜ, Mitchell Marta-Arizaª, Jose A Chabalgoityᶜ, Thomas Wisniewskiª,>d

CONTACT Fernando Goñi Fernando.goni@nyumc.org

## ABSTRACT

**Background/Introduction:** Transmissible prion diseases have become a worldwide problem of enormous proportions with no prevention or cure in sight. The North American and northern Europe Chronic Wasting disease (CWD) epidemic in free range moose, elk and deer has not been contained. We were successful in developing an oral vaccination with PrP molecules delivered by an attenuated Salmonella carrier to prevent transmission and infection of susceptible mice, and achieve, in white-tailed deer, partial mucosal and systemic antibody protection to an oral challenge with an infective CWD-prion [1,2]. We have now refined the constructs to small peptides with specificities for multiple mammalian PrP molecules for a more effective oral vaccine.

**Materials and Methods:** We designed and constructed 20–30 residues long multi-species PrP-like peptides with amino acids specific for at least three different species and encompassing the whole PrP molecule. Peptides were expressed in an attenuated Salmonella delivery system, or controlled glutaraldehyde polymerized with and without cholera toxin-B (CTB). Vaccine preparations were kept for 7 days at 4°C and then for 36 h at RT. All preparations were inoculated by gavage into mice transgenic for different species PrP. After three weekly inoculations antibody production was measured in faeces and serum.

**Results:** Viability of the vaccines after 10 days was between 87% and 95%. Using a reduced number of inoculations, all Tg CD-1 mice expressing human, elk, sheep or mouse PrP and PrP-KO produced a sustained antibody response (both IgA and IgG) that cross-reacted with prion proteins from different species.

**Conclusions:** Stability and ease of production of these new vaccine formulations would allow for up scaling and use in larger animals of any species administered as food supplement in enclosed facilities or potentially as bait in the wild. The cross-reactivity of the antibodies produced would help neutralize any infective prion from oral transmission and inhibit spread of the β-sheet structure responsible for $PrP^C$ to $PrP^{Sc}$ conversion [3].

**KEYWORDS:** CWD; pan-vaccine; multi-species; oral vaccine

## References

[1] Goñi, et al. Neuroscience 2008; 153:679686.

[2] Goñi, et al. Vaccine 2015; 33:726733.

[3] Wisniewski and Goñi. Handbook of Clinical Neurology 2018; 153:419–430.

<footnote>ªDepartment of Neurology New York University, NY, USA; ᵇINTA, Buenos Aires Argentina; ᵇBiotech Department of Universidad de la Republica, Uruguay; ᵈDepts of Pathology and Psychiatry NYUSoM, USA</footnote>
<figure><img src="image_103.png" /></figure>

order to understand the diversity of goat TSE strains and to discriminate BSE from them. Here, we studied goat TSEs by their relative transmission efficiency in different animal models and by the $PrP^{Sc}$ type(s) propagated in rodents. Thus, the biological properties of TSE isolates were inferred by their specific interaction with different recipient PrP species rather than by serial passage in a single model. Goat TSE isolates from seven countries and experimental goat-BSE were inoculated in seven models: tg-mice overexpressing sheep/goat ARQ, sheep VRQ, cattle or mouse PrPs; RIII mice and bank voles. The attack rate and the survival time were combined to obtain the 'transmission efficiency', a parameter used to build the 'transmission profiles' of individual isolates across all models. Goat-BSE and Nor98 showed distinctive transmission profiles, different among them and from all other isolates. Goat-BSE was the only isolate inducing 19kDa $PrP^{Sc}$ in all rodent models, while Nor98 was associated with 8kDa $PrP^{Sc}$. CS isolates showed a strong variability of transmission profiles, which was in part explained by their geographical distribution, and could be grouped into four distinct categories. CS isolates in category 1 induced the propagation solely of 21kDa $PrP^{Sc}$, while isolates from CS categories 2-to-4 also induced, to a different extent depending on the model, a 19kDa $PrP^{Sc}$ type distinct from BSE. Further investigation of CNS and LRS tissues from a goat herd showed that this phenomenon reflects the selective amplification in rodents of at least two co-existing 'sub-strain components', 21kDa and 19kDa, whose relative amount varies with different goat tissues/isolates. Our findings show that BSE can be discriminated from a wide range of biologically and geographically diverse goat TSEs. The geographical variation observed in CS suggests the existence of distinct CS strains, as also supported by the parallel studies by Pirisinu et al. and Marin-Moreno et al. Yet, CS isolates in categories 2-to-4 were mixtures of discrete sub-strains, where it could be envisaged that competition among sub-strains play a role in defining the biological properties of individual isolates and their evolution.

## 162. 'Multi-species anti-prion oral vaccine for familial and transmissible spongiform encephalopathies'

Fernando Goñiª, Analia Eliseiᵇ, Lucia Yimᶜ, Mitchell Marta-Arizaª, Jose A Chabalgoityᶜ, Thomas Wisniewskiª,>d

CONTACT Fernando Goñi Fernando.goni@nyumc.org

## ABSTRACT

**Background/Introduction:** Transmissible prion diseases have become a worldwide problem of enormous proportions with no prevention or cure in sight. The North American and northern Europe Chronic Wasting disease (CWD) epidemic in free range moose, elk and deer has not been contained. We were successful in developing an oral vaccination with PrP molecules delivered by an attenuated Salmonella carrier to prevent transmission and infection of susceptible mice, and achieve, in white-tailed deer, partial mucosal and systemic antibody protection to an oral challenge with an infective CWD-prion [1,2]. We have now refined the constructs to small peptides with specificities for multiple mammalian PrP molecules for a more effective oral vaccine.

**Materials and Methods:** We designed and constructed 20–30 residues long multi-species PrP-like peptides with amino acids specific for at least three different species and encompassing the whole PrP molecule. Peptides were expressed in an attenuated Salmonella delivery system, or controlled glutaraldehyde polymerized with and without cholera toxin-B (CTB). Vaccine preparations were kept for 7 days at 4°C and then for 36 h at RT. All preparations were inoculated by gavage into mice transgenic for different species PrP. After three weekly inoculations antibody production was measured in faeces and serum.

**Results:** Viability of the vaccines after 10 days was between 87% and 95%. Using a reduced number of inoculations, all Tg CD-1 mice expressing human, elk, sheep or mouse PrP and PrP-KO produced a sustained antibody response (both IgA and IgG) that cross-reacted with prion proteins from different species.

**Conclusions:** Stability and ease of production of these new vaccine formulations would allow for up scaling and use in larger animals of any species administered as food supplement in enclosed facilities or potentially as bait in the wild. The cross-reactivity of the antibodies produced would help neutralize any infective prion from oral transmission and inhibit spread of the β-sheet structure responsible for $PrP^C$ to $PrP^{Sc}$ conversion [3].

**KEYWORDS:** CWD; pan-vaccine; multi-species; oral vaccine

## References

[1] Goñi, et al. Neuroscience 2008; 153:679686.

[2] Goñi, et al. Vaccine 2015; 33:726733.

[3] Wisniewski and Goñi. Handbook of Clinical Neurology 2018; 153:419–430.

<footnote>ªDepartment of Neurology New York University, NY, USA; ᵇINTA, Buenos Aires Argentina; ᵇBiotech Department of Universidad de la Republica, Uruguay; ᵈDepts of Pathology and Psychiatry NYUSoM, USA</footnote>
<figure><img src="image_104.png" /></figure>

**163. Structural characterization of α-synuclein prions via cryo-electron microscopy**

Gregory E. Merzª, Eric Tseª, Victor Banerjeeª, Amanda L. Woermanª,ᵇ, William F. DeGradoª,ᵈ, Daniel R. Southworthª,ᶜ and Stanley B. Prusinerª,ᵇ,ᶜ

ªInstitute for Neurodegenerative Diseases, Weill Institute for Neurosciences, University of California, San Francisco, CA, USA;
ᵇDepartment of Neurology, University of California, San Francisco, CA, USA;
ᶜDepartment of Biochemistry and Biophysics, University of California, San Francisco, CA, USA;
ᵈDepartment of Pharmaceutical Chemistry, Cardiovascular Research Institute, University of California, San Francisco, CA, USA

CONTACT Gregory E. Merz gregory.merz@ucsf.edu

**ABSTRACT**

Recent structural characterization via cryo-electron microscopy (cryo-EM) has confirmed at high-resolution that different fibrillar structures of tau prions (or tau strains) exhibit clinically distinct tauopathy disorders. Such findings are consistent with the hypothesis that the biological properties of prion strains are determined by their misfolded conformations. Similarly, several groups have recently demonstrated that the synucleinopathies of Parkinson's disease (PD) and multiple system atrophy (MSA) are caused by α-synuclein aggregates with distinct biological activities. Given that the structure of prions determines their biological properties, we hypothesize that α-synuclein prions in PD and MSA have unique fibrillar conformations. We are investigating this structural hypothesis by characterizing α-synuclein prions at high resolution using cryo-EM. Several strains of α-synuclein prions will be purified from human brain as well as homogenates passaged through Tg mice and cultured cells. We will perform cryo-EM on proteins purified from brain homogenates to obtain the high-resolution information necessary to make structural comparisons between strains at the molecular level. The overall folds of prions, as well as inter- and intra-molecular interactions, will be evaluated across prion strains. Mutation sites in α-synuclein causing familial synucleinopathies (e.g. A53T) are of particular interest, and the interactions of these residues within the larger fibrillar fold will be closely studied. The unequivocal assignment of specific α-synuclein conformations with the resulting disease is likely to represent a major step forward in our understanding of the pathogenesis of synucleinopathies.

**164. Overview of scrapie cases in Iceland 2004–2018**

Eva Hauksdottir and Stefania Thorgeirdsottir

Keldur, the Institute for Experimental Pathology, University of Iceland

CONTACT Eva Hauksdottir evahauks@hi.is

**ABSTRACT**

**Introduction:** Since 1978 Iceland has actively screened healthy slaughtered (HS) sheep for scrapie, however, in 2004 the screening method changed from histopathological staining to a rapid ELISA test. With this new method scrapie cases in HS were identified for the first time, as before index scrapie cases had only been detected in sheep with clinical symptoms (CS). Use of this sensitive method also led to the detection of the first atypical scrapie case (Nor98) in Iceland in 2004.

**Materials and Methods:** During 2004–2018 there were 50,515 HS samples tested with ELISA, along with 558 samples from fallen stock and clinical suspects. In this overview, the cases detected during this time, will be analysed further, e.g. origin, age, PrP genotype and number of additional cases from each farm detected after culling of the scrapie flock.

**Results:** In the years 2004–2018 there were 29 scrapie cases identified, where one case refers to a single affected farm. Two thirds of the classical scrapie cases originate from CS sheep, while Nor98 is generally identified in HS samples rather than CS. The genotype most associated with susceptibility to classical scrapie is frequent in the classical scrapie cases, however a neutral genotype is found in many of those cases as well. After culling, where a scrapie flock (or a portion of it) was tested, additional positive samples of classical scrapie were frequently detected, but only once has an additional Nor98 case been found in a culled flock.

**Conclusion:** The comparison of classical and atypical scrapie in Iceland shows that they have many different qualities in relation to origin, symptoms, genotype and flock infectivity.

**165. Familial Parkinson's point mutation abolishes multiple system atrophy prion replication**

Amanda L. Woermanª,ᵇ, Sabeen Kazmiª, Smita Patelª, Abby Oehlerª, Kartika Widjajaª, Daniel A. Mordesᶜ, Steven H. Olsonª,ᵇ and Stanley B. Prusinerª,ᵇ,ᵈ

ªInstitute for Neurodegenerative Diseases, Weill Institute for Neurosciences, University of California, San Francisco, CA, USA;
ᵇDepartment of Neurology, University of California, San Francisco, CA, USA;
ᶜC.S. Kubik Laboratory for Neuropathology, Department of Pathology, Massachusetts General Hospital, Boston, MA, USA;
ᵈDepartment of Biochemistry and Biophysics, University of California, San Francisco, CA, USA

CONTACT Amanda L. Woerman amanda.woerman@ucsf.edu
<figure><img src="image_104.png" /></figure>

# 163. Structural characterization of α-synuclein prions via cryo-electron microscopy

Gregory E. Merzª, Eric Tseª, Victor Banerjeeª, Amanda L. Woermanª,ᵇ, William F. DeGradoª,ᵈ, Daniel R. Southworthª,ᶜ and Stanley B. Prusinerª,ᵇ,ᶜ

ªInstitute for Neurodegenerative Diseases, Weill Institute for Neurosciences, University of California, San Francisco, CA, USA; ᵇDepartment of Neurology, University of California, San Francisco, CA, USA; ᶜDepartment of Biochemistry and Biophysics, University of California, San Francisco, CA, USA; ᵈDepartment of Pharmaceutical Chemistry, Cardiovascular Research Institute, University of California, San Francisco, CA, USA

CONTACT Gregory E. Merz gregory.merz@ucsf.edu

## ABSTRACT

Recent structural characterization via cryo-electron microscopy (cryo-EM) has confirmed at high-resolution that different fibrillar structures of tau prions (or tau strains) exhibit clinically distinct tauopathy disorders. Such findings are consistent with the hypothesis that the biological properties of prion strains are determined by their misfolded conformations. Similarly, several groups have recently demonstrated that the synucleinopathies of Parkinson's disease (PD) and multiple system atrophy (MSA) are caused by α-synuclein aggregates with distinct biological activities. Given that the structure of prions determines their biological properties, we hypothesize that α-synuclein prions in PD and MSA have unique fibrillar conformations. We are investigating this structural hypothesis by characterizing α-synuclein prions at high resolution using cryo-EM. Several strains of α-synuclein prions will be purified from human brain as well as homogenates passaged through Tg mice and cultured cells. We will perform cryo-EM on proteins purified from brain homogenates to obtain the high-resolution information necessary to make structural comparisons between strains at the molecular level. The overall folds of prions, as well as inter- and intra-molecular interactions, will be evaluated across prion strains. Mutation sites in α-synuclein causing familial synucleinopathies (e.g. A53T) are of particular interest, and the interactions of these residues within the larger fibrillar fold will be closely studied. The unequivocal assignment of specific α-synuclein conformations with the resulting disease is likely to represent a major step forward in our understanding of the pathogenesis of synucleinopathies.

# 164. Overview of scrapie cases in Iceland 2004–2018

Eva Hauksdottir and Stefania Thorgeirdsottir

Keldur, the Institute for Experimental Pathology, University of Iceland

CONTACT Eva Hauksdottir evahauks@hi.is

## ABSTRACT

**Introduction:** Since 1978 Iceland has actively screened healthy slaughtered (HS) sheep for scrapie, however, in 2004 the screening method changed from histopathological staining to a rapid ELISA test. With this new method scrapie cases in HS were identified for the first time, as before index scrapie cases had only been detected in sheep with clinical symptoms (CS). Use of this sensitive method also led to the detection of the first atypical scrapie case (Nor98) in Iceland in 2004.

**Materials and Methods:** During 2004–2018 there were 50,515 HS samples tested with ELISA, along with 558 samples from fallen stock and clinical suspects. In this overview, the cases detected during this time, will be analysed further, e.g. origin, age, PrP genotype and number of additional cases from each farm detected after culling of the scrapie flock.

**Results:** In the years 2004–2018 there were 29 scrapie cases identified, where one case refers to a single affected farm. Two thirds of the classical scrapie cases originate from CS sheep, while Nor98 is generally identified in HS samples rather than CS. The genotype most associated with susceptibility to classical scrapie is frequent in the classical scrapie cases, however a neutral genotype is found in many of those cases as well. After culling, where a scrapie flock (or a portion of it) was tested, additional positive samples of classical scrapie were frequently detected, but only once has an additional Nor98 case been found in a culled flock.

**Conclusion:** The comparison of classical and atypical scrapie in Iceland shows that they have many different qualities in relation to origin, symptoms, genotype and flock infectivity.

# 165. Familial Parkinson's point mutation abolishes multiple system atrophy prion replication

Amanda L. Woermanª,ᵇ, Sabeen Kazmiª, Smita Patelª, Abby Oehlerª, Kartika Widjajaª, Daniel A. Mordesᶜ, Steven H. Olsonª,ᵇ and Stanley B. Prusinerª,ᵇ,ᵈ

CONTACT Amanda L. Woerman amanda.woerman@ucsf.edu

<footnote>ªInstitute for Neurodegenerative Diseases, Weill Institute for Neurosciences, University of California, San Francisco, CA, USA; ᵇDepartment of Neurology, University of California, San Francisco, CA, USA; ᶜC.S. Kubik Laboratory for Neuropathology, Department of Pathology, Massachusetts General Hospital, Boston, MA, USA; ᵈDepartment of Biochemistry and Biophysics, University of California, San Francisco, CA, USA</footnote>
<figure><img src="image_105.png" /></figure>

**ABSTRACT**

In the neurodegenerative disease multiple system atrophy (MSA), α-synuclein misfolds into a self-templating conformation to become a prion. To compare the biological activity of α-synuclein prions in MSA and Parkinson's disease (PD), we developed nine α-synuclein-YFP cell lines expressing point mutations responsible for inherited PD. MSA prions robustly infected wild-type, A30P, and A53T α-synuclein-YFP cells, but they were unable to replicate in cells expressing the E46K mutation. Coexpression of the A53T and E46K mutations was unable to rescue MSA prion infection *in vitro*, establishing that MSA α-synuclein prions are conformationally distinct from the misfolded α-synuclein in PD patients. This observation may have profound implications for developing treatments for neurodegenerative diseases.

**166. α-cleavage of PrPC by Aspirin suppresses prion infection**

Huyen Trang Trinhª, Taeyeon Kimᵇ, A-Ran Kimª, Junwoo Shinª, Hakmin Leeª, Jieun Kimª, Sungeun Leeª, Chongsuk Ryouª

CONTACT Huyen Trang Trinh ds.trinhthihuyentrang@gmail.com; Chongsuk Ryou cryou2@hanyang.ac.kr

**ABSTRACT**

**Background:** The conversion of the cellular prion protein, PrPC, into its pathogenic form, PrPSc, is the hallmark of prion diseases. In physiological conditions, PrPC can be cleaved at α-site, which generates the C1 and N1 fragments, and disrupts a region critical for conversion into PrPSc. Plasmin is known as an enzyme that cleaves PrPC at α-site; however, the function of plasmin in prion propagation remains elusive. In the current study, the role of plasmin and Aspirin, known to stimulate plasmin activity, in α-cleavage of PrPC and prion suppression was investigated.

**Material and Methods:** The effect of plasmin and Aspirin was evaluated using an in vitro assay, which mimics the aggregation process of prion protein into amyloids. In the cell-based model, ScN2a cells were incubated with Aspirin and endogenous plasmin activity was measured. The generation of C1 and N1 fragments were also detected by immunoblotting. The elimination of PrPSc by Aspirin was investigated in ScN2a. Finally, the ability of Aspirin to interfere with

the susceptibility of N2a to prions was determined using standard scrapie cell assay.

**Results:** Aspirin promoted plasmin activity by inducing α-cleavage of PrP, resulting in the inhibition of PrP aggregation in vitro. Moreover, the endogenous plasmin activity in the ScN2a was significantly enhanced by the action of Aspirin. The generation of C1 fragment was increased in the membrane fraction of the cells incubated with Aspirin, indicating that Aspirin activated α-cleavage of PrPC. In addition, Aspirin significantly reduced the level of PrPSc in cells permanently infected with prions. Finally, Aspirin significantly decreased the number of N2a cells infected with prions.

**Conclusions:** Aspirin promoting plasmin-mediated α-cleavage of PrPC suppressed PrPSc propagation.

**KEYWORDS:** Aspirin; plasmin; α-cleavage; C1 fragment

**167. Two different conformers of type 1 prion protein propagate as distinct strains in transgenic mice**

Ignazio Caliª,ᵇ,\* Juan Carlos Espinosaᶜ,\*, Tetsuyuki Kitamotoᵈ, Alba Marin-Morenoᶜ, Brian S. Applebyª,ᵇ,ᵉ, Juan Maria Torresᶜ and Pierluigi Gambettiª

CONTACT Ignazio Cali ignazio.cali@case.edu

\*These authors contributed equally to this work

**ABSTRACT**

A modern classification recognizes five subtypes of sporadic Creutzfeldt-Jakob disease (sCJD)-based on the pairing of methionine (M)/valine (V) polymorphic genotype at codon 129 of the prion protein (PrP), which determines the MM, MV and VV 129 genotypes, and the type 1 or 2 of the disease-associated PrP (PrP<sup>D</sup>)<sup>1</sup>. These two PrP<sup>D</sup> types are distinguished on the basis of their different electrophoretic mobility as the PK-resistant PrP<sup>D</sup> (resPrP<sup>D</sup>) type 1 and type 2 migrate to ~21 and ~19 kDa, respectively. In sCJDVV1, the electrophoretic profile of resPrP<sup>D</sup> is distinct from that of resPrP<sup>D</sup> type 1 associated with the sCJDMM (MV)1 subtype. It features a heterogeneous component, which in its unglycosylated form appears in three versions: a single band of ~20 kDa (T1<sup>20</sup>), of ~21 kDa (T1<sup>21</sup>) or as a

<footnote>ªDepartment of Pharmacy, College of Pharmacy and Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan, Gyeonggi-do, Republic of Korea; ᵇDivision of Developmental Biology and Physiology, School of Biosciences and Chemistry, Institute for Basic Science, Sungshin University, Seoul, Republic of Korea</footnote>

<footnote>ªDepartment of Pathology and ᵇNational Prion Disease Pathology Surveillance Center (NPDPSC), Case Western Reserve University, School of Medicine, Cleveland, OH 44,106, USA; ᶜCentro de Investigación en Sanidad Animal, CISA-INIA, Madrid, Spain; ᵈDepartment of Neurological Science, Tohoku University Graduate School of Medicine, Sendai, Japan; ᵉDepartment of Neurology, University Hospitals Cleveland Medical Center, Beachwood, OH 44,122, USA.</footnote>
<figure><img src="image_105.png" /></figure>

**ABSTRACT**

In the neurodegenerative disease multiple system atrophy (MSA), α-synuclein misfolds into a self-templating conformation to become a prion. To compare the biological activity of α-synuclein prions in MSA and Parkinson's disease (PD), we developed nine α-synuclein-YFP cell lines expressing point mutations responsible for inherited PD. MSA prions robustly infected wild-type, A30P, and A53T α-synuclein-YFP cells, but they were unable to replicate in cells expressing the E46K mutation. Coexpression of the A53T and E46K mutations was unable to rescue MSA prion infection *in vitro*, establishing that MSA α-synuclein prions are conformationally distinct from the misfolded α-synuclein in PD patients. This observation may have profound implications for developing treatments for neurodegenerative diseases.

**166. α-cleavage of PrPC by Aspirin suppresses prion infection**

Huyen Trang Trinhª, Taeyeon Kimᵇ, A-Ran Kimª, Junwoo Shinª, Hakmin Leeª, Jieun Kimª, Sungeun Leeª, Chongsuk Ryouª

CONTACT Huyen Trang Trinh ds.trinhthihuyentrang@gmail.com; Chongsuk Ryou cryou2@hanyang.ac.kr

**ABSTRACT**

**Background:** The conversion of the cellular prion protein, PrPC, into its pathogenic form, PrPSc, is the hallmark of prion diseases. In physiological conditions, PrPC can be cleaved at α-site, which generates the C1 and N1 fragments, and disrupts a region critical for conversion into PrPSc. Plasmin is known as an enzyme that cleaves PrPC at α-site; however, the function of plasmin in prion propagation remains elusive. In the current study, the role of plasmin and Aspirin, known to stimulate plasmin activity, in α-cleavage of PrPC and prion suppression was investigated.

**Material and Methods:** The effect of plasmin and Aspirin was evaluated using an in vitro assay, which mimics the aggregation process of prion protein into amyloids. In the cell-based model, ScN2a cells were incubated with Aspirin and endogenous plasmin activity was measured. The generation of C1 and N1 fragments were also detected by immunoblotting. The elimination of PrPSc by Aspirin was investigated in ScN2a. Finally, the ability of Aspirin to interfere with

the susceptibility of N2a to prions was determined using standard scrapie cell assay.

**Results:** Aspirin promoted plasmin activity by inducing α-cleavage of PrP, resulting in the inhibition of PrP aggregation in vitro. Moreover, the endogenous plasmin activity in the ScN2a was significantly enhanced by the action of Aspirin. The generation of C1 fragment was increased in the membrane fraction of the cells incubated with Aspirin, indicating that Aspirin activated α-cleavage of PrPC. In addition, Aspirin significantly reduced the level of PrPSc in cells permanently infected with prions. Finally, Aspirin significantly decreased the number of N2a cells infected with prions.

**Conclusions:** Aspirin promoting plasmin-mediated α-cleavage of PrPC suppressed PrPSc propagation.

**KEYWORDS:** Aspirin; plasmin; α-cleavage; C1 fragment

**167. Two different conformers of type 1 prion protein propagate as distinct strains in transgenic mice**

Ignazio Caliª,ᵇ,\* Juan Carlos Espinosaᶜ,\*, Tetsuyuki Kitamotoᵈ, Alba Marin-Morenoᶜ, Brian S. Applebyª,ᵇ,ᵉ, Juan Maria Torresᶜ and Pierluigi Gambettiª

CONTACT Ignazio Cali ignazio.cali@case.edu

\*These authors contributed equally to this work

**ABSTRACT**

A modern classification recognizes five subtypes of sporadic Creutzfeldt-Jakob disease (sCJD)-based on the pairing of methionine (M)/valine (V) polymorphic genotype at codon 129 of the prion protein (PrP), which determines the MM, MV and VV 129 genotypes, and the type 1 or 2 of the disease-associated PrP (PrP<sup>D</sup>)<sup>1</sup>. These two PrP<sup>D</sup> types are distinguished on the basis of their different electrophoretic mobility as the PK-resistant PrP<sup>D</sup> (resPrP<sup>D</sup>) type 1 and type 2 migrate to ~21 and ~19 kDa, respectively. In sCJDVV1, the electrophoretic profile of resPrP<sup>D</sup> is distinct from that of resPrP<sup>D</sup> type 1 associated with the sCJDMM (MV)1 subtype. It features a heterogeneous component, which in its unglycosylated form appears in three versions: a single band of ~20 kDa (T1<sup>20</sup>), of ~21 kDa (T1<sup>21</sup>) or as a

<footnote>ªDepartment of Pharmacy, College of Pharmacy and Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan, Gyeonggi-do, Republic of Korea; ᵇDivision of Developmental Biology and Physiology, School of Biosciences and Chemistry, Institute for Basic Science, Sungshin University, Seoul, Republic of Korea</footnote>

<footnote>ªDepartment of Pathology and ᵇNational Prion Disease Pathology Surveillance Center (NPDPSC), Case Western Reserve University, School of Medicine, Cleveland, OH 44,106, USA; ᶜCentro de Investigación en Sanidad Animal, CISA-INIA, Madrid, Spain; ᵈDepartment of Neurological Science, Tohoku University Graduate School of Medicine, Sendai, Japan; ᵉDepartment of Neurology, University Hospitals Cleveland Medical Center, Beachwood, OH 44,122, USA.</footnote>
<figure><img src="image_106.png" /></figure>

doublet with both ~20 and ~21 kDa bands (T1²⁰⁻²¹). Although these bands are a minor component resPrP<sup>D</sup>, their consistent presence raises the issue as to whether they are a feature that distinguish the PrP<sup>D</sup> strain associated with sCJDVV1 from that associated with sCJDMM(MV)1 and other type 1 strains. We have carried out a retrospective examination of brain homogenates from subjects with the definitive diagnosis of sCJDVV1 that were collected at the National Prion Disease Pathology Surveillance Center (NPDPSC). Western blot (WB) analysis confirmed the presence of the three distinct electrophoretic profiles of unglycosylated resPrP<sup>D</sup>. To determine whether these distinct molecular signatures of resPrP<sup>D</sup> associated with sCJDVV1 could be propagated *in vivo*, brain homogenates from sCJDVV1 cases harbouring each of the three profiles were inoculated to transgenic (Tg) mice expressing human PrP-129V. The T1²⁰ and T1²¹ WB profiles were faithfully replicated in the Tg mice. Although as in sCJDVV1 they were associated with undistinguishable histopathological phenotypes the incubation periods following T1²⁰ and T1²¹ inoculations differed. Moreover, similar inoculations to Tg mice expressing PrP-129M, i.e. mismatched at codon 129 with the PrP-codon 129 of the inoculum, hampered transmission of T1²¹ and delayed that of T1²⁰. Combined, these data indicate that T1²¹ and T1²⁰ represent two distinct human prion substrains which however are not able to determine distinct phenotypes.

## Funding

Supported by National Institutes of Health Grants R01 NS083687 and P01 AI106705, and The Charles S. Britton Fund (to P.G.). This study was also supported by a grant from the Spanish Ministerio de Ciencia, Innovación y Universidades (AGL2016-78054-R - AEI/FEDER, UE).

## References

[1] Parchi et al. Ann Neurol. 1999;46(2):224-233.

[2] Parchi et al. Acta Neuropathol. 2011;121:91-112

## **168. Clinical prediction of type 1 and type 2 mixed histopathology distribution in MM-type sporadic Creutzfeldt-Jakob disease**

Toshimasa Ikeda<sup>a,b</sup>, Yasushi Iwasaki<sup>a</sup>, Keita Sakurai<sup>c</sup>, Akio Akagi<sup>a</sup>, Maya Mimuro<sup>a</sup>, Hiroaki Miyahara<sup>a</sup>, Tetsuyuki Kitamoto<sup>d</sup>, Noriyuki Matsukawa<sup>b</sup>, Mari Yoshida<sup>a</sup>

CONTACT Toshimasa Ikeda c161706@ed.nagoya-cu.ac.jp

## ABSTRACT

**Background/Introduction:** Although the sporadic Creutzfeldt-Jakob disease (sCJD) subtypes, with their distinctive clinicopathological features, have been identified based on two types of the abnormal prion protein (PrP<sup>Sc</sup>), type 1 and type 2, and polymorphic codon 129, there have been cases of both PrP<sup>Sc</sup> types being present. The mixed PrP<sup>Sc</sup> type features, especially in MM-type CJD, have been best identified with neuropathological examination; the MM1-type involves fine vacuole-type spongiform change (FV), whereas the MM2C-type involves large confluent vacuole-type spongiform change (LCV). Presently, it is impossible to detect the concurrence of both PrP<sup>Sc</sup> types before death. The purpose of this study was to clinically predict the concurrence of MM-type sCJD with another PrP<sup>Sc</sup> type in the same individual.

**Materials and Methods:** We retrospectively identified seven MM-type sCJD cases with both FV and LCV among 49 sCJD cases genetico-pathologically diagnosed in our institute between 2008 and 2016. We reviewed clinical features, pathological findings, and radiological abnormalities. We also conducted a regional systemic study to associate the spongiform change pattern with hyperintensity on magnetic resonance diffusion-weighted imaging (DWI) using the signal intensity index (SII).

**Results:** The one patient with dominant LCV showed longer disease duration, later onset of typical symptoms, no periodic sharp wave complexes in electroencephalography, and negative 14-3-3 protein findings compared to the six FV-dominant patients. LCV-dominant lesions tended to show higher intensity on DWI than FV-dominant lesions in respective patients. In the regional systemic study, LCV-dominant regions showed significantly higher SII on DWI than did the FV-dominant regions.

**Conclusions:** Mixed MM-type sCJD showed clinical features correlating with the dominant pathological burden. The SII may be clinically useful for investigating the concurrence of PrP<sup>Sc</sup> type 2 in cases with the typical clinical course of MM1-type sCJD.

**KEYWORDS:** Creutzfeldt-Jakob disease; histopathology; diffusion-weighted imaging (DWI); magnetic resonance imaging (MRI); spongiform change

## **169. Stable propagation of hamster prions in CRISPR/Cas9-engineered CAD5 cells**

Matthew Bourkas<sup>a,b</sup>, Hamza Arshad<sup>a,b</sup>, Gerold Schmitt-Ulms<sup>a,c</sup>, Jason Bartz<sup>d</sup> and Joel Watts<sup>a,b</sup>

<footnote><sup>a</sup>Department of Neuropathology, Institute for Medical Science of Aging, Aichi Medical University, Nagakute, Japan; <sup>b</sup>Department of Neurology and Neuroscience, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan; <sup>c</sup>Department of Radiology, Teikyo University School of Medicine, Tokyo, Japan;</footnote>

<footnote><sup>d</sup>Department of Neurological Science, Tohoku University Graduate School of Medicine, Sendai, Japan</footnote>
<figure><img src="image_106.png" /></figure>

doublet with both ~20 and ~21 kDa bands (T1²⁰⁻²¹). Although these bands are a minor component resPrP<sup>D</sup>, their consistent presence raises the issue as to whether they are a feature that distinguish the PrP<sup>D</sup> strain associated with sCJDVV1 from that associated with sCJDMM(MV)1 and other type 1 strains. We have carried out a retrospective examination of brain homogenates from subjects with the definitive diagnosis of sCJDVV1 that were collected at the National Prion Disease Pathology Surveillance Center (NPDPSC). Western blot (WB) analysis confirmed the presence of the three distinct electrophoretic profiles of unglycosylated resPrP<sup>D</sup>. To determine whether these distinct molecular signatures of resPrP<sup>D</sup> associated with sCJDVV1 could be propagated *in vivo*, brain homogenates from sCJDVV1 cases harbouring each of the three profiles were inoculated to transgenic (Tg) mice expressing human PrP-129V. The T1²⁰ and T1²¹ WB profiles were faithfully replicated in the Tg mice. Although as in sCJDVV1 they were associated with undistinguishable histopathological phenotypes the incubation periods following T1²⁰ and T1²¹ inoculations differed. Moreover, similar inoculations to Tg mice expressing PrP-129M, i.e. mismatched at codon 129 with the PrP-codon 129 of the inoculum, hampered transmission of T1²¹ and delayed that of T1²⁰. Combined, these data indicate that T1²¹ and T1²⁰ represent two distinct human prion substrains which however are not able to determine distinct phenotypes.

## Funding

Supported by National Institutes of Health Grants R01 NS083687 and P01 AI106705, and The Charles S. Britton Fund (to P.G.). This study was also supported by a grant from the Spanish Ministerio de Ciencia, Innovación y Universidades (AGL2016-78054-R - AEI/FEDER, UE).

## References

[1] Parchi et al. Ann Neurol. 1999;46(2):224-233.

[2] Parchi et al. Acta Neuropathol. 2011;121:91-112

## 168. Clinical prediction of type 1 and type 2 mixed histopathology distribution in MM-type sporadic Creutzfeldt-Jakob disease

Toshimasa Ikeda<sup>a,b</sup>, Yasushi Iwasaki<sup>a</sup>, Keita Sakurai<sup>c</sup>, Akio Akagi<sup>a</sup>, Maya Mimuro<sup>a</sup>, Hiroaki Miyahara<sup>a</sup>, Tetsuyuki Kitamoto<sup>d</sup>, Noriyuki Matsukawa<sup>b</sup>, Mari Yoshida<sup>a</sup>

CONTACT Toshimasa Ikeda c161706@ed.nagoya-cu.ac.jp

## ABSTRACT

**Background/Introduction:** Although the sporadic Creutzfeldt-Jakob disease (sCJD) subtypes, with their distinctive clinicopathological features, have been identified based on two types of the abnormal prion protein (PrP<sup>Sc</sup>), type 1 and type 2, and polymorphic codon 129, there have been cases of both PrP<sup>Sc</sup> types being present. The mixed PrP<sup>Sc</sup> type features, especially in MM-type CJD, have been best identified with neuropathological examination; the MM1-type involves fine vacuole-type spongiform change (FV), whereas the MM2C-type involves large confluent vacuole-type spongiform change (LCV). Presently, it is impossible to detect the concurrence of both PrP<sup>Sc</sup> types before death. The purpose of this study was to clinically predict the concurrence of MM-type sCJD with another PrP<sup>Sc</sup> type in the same individual.

**Materials and Methods:** We retrospectively identified seven MM-type sCJD cases with both FV and LCV among 49 sCJD cases genetico-pathologically diagnosed in our institute between 2008 and 2016. We reviewed clinical features, pathological findings, and radiological abnormalities. We also conducted a regional systemic study to associate the spongiform change pattern with hyperintensity on magnetic resonance diffusion-weighted imaging (DWI) using the signal intensity index (SII).

**Results:** The one patient with dominant LCV showed longer disease duration, later onset of typical symptoms, no periodic sharp wave complexes in electroencephalography, and negative 14-3-3 protein findings compared to the six FV-dominant patients. LCV-dominant lesions tended to show higher intensity on DWI than FV-dominant lesions in respective patients. In the regional systemic study, LCV-dominant regions showed significantly higher SII on DWI than did the FV-dominant regions.

**Conclusions:** Mixed MM-type sCJD showed clinical features correlating with the dominant pathological burden. The SII may be clinically useful for investigating the concurrence of PrP<sup>Sc</sup> type 2 in cases with the typical clinical course of MM1-type sCJD.

**KEYWORDS:** Creutzfeldt-Jakob disease; histopathology; diffusion-weighted imaging (DWI); magnetic resonance imaging (MRI); spongiform change

## 169. Stable propagation of hamster prions in CRISPR/Cas9-engineered CAD5 cells

Matthew Bourkas<sup>a,b</sup>, Hamza Arshad<sup>a,b</sup>, Gerold Schmitt-Ulms<sup>a,c</sup>, Jason Bartz<sup>d</sup> and Joel Watts<sup>a,b</sup>

<footnote><sup>a</sup>Department of Neuropathology, Institute for Medical Science of Aging, Aichi Medical University, Nagakute, Japan; <sup>b</sup>Department of Neurology and Neuroscience, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan; <sup>c</sup>Department of Radiology, Teikyo University School of Medicine, Tokyo, Japan;</footnote>

<footnote><sup>d</sup>Department of Neurological Science, Tohoku University Graduate School of Medicine, Sendai, Japan</footnote>
<figure><img src="image_107.png" /></figure>

<sup>a</sup>Tanz Centre for Research in Neurodegenerative Diseases, Toronto, Canada; <sup>b</sup>Department of Biochemistry, University of Toronto, Toronto, Canada; <sup>c</sup>Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada 4 Department of Medical Microbiology and Immunology, Creighton University, Omaha, USA

CONTACT Matthew Bourkas matthew.bourkas@mail.utoronto.ca

## ABSTRACT

**Background:** Prion research has been hindered by a lack of cellular paradigms for studying the replication of prions from different species. Although hamster prions have been widely used to study prion replication in animals and within *in vitro* amplification systems, they have proven challenging to propagate in cultured cells. Since the murine catecholaminergic cell line CAD5 is uniquely susceptible to a diverse range of mouse prion strains, [1,2] we hypothesized that it might also be capable of propagating non-mouse prions.

**Materials and Methods:** We generated CAD5 cells lacking endogenous PrP<sup>C</sup> (CAD5-PrP<sup>-/-</sup> cells) using CRISPR/Cas9-mediated genome editing. CAD5-PrP<sup>-/-</sup> cells were stably transfected with a plasmid encoding hamster PrP and then challenged with brain homogenates containing various strains of hamster prions.

**Results:** When exposed to the 263K, HY, or 139H strains, hamster PrP-expressing CAD5-PrP<sup>-/-</sup> cells stably propagated high levels of protease-resistant PrP. Moreover, when these cells were challenged with 10-fold serial dilutions of 263K prions, PK-resistant PrP was obtained with dilutions up to and including 0.000002% brain homogenate. Cellular homogenates from 263K-infected cells exhibited prion seeding activity in the RT-QuIC assay and were infectious to naïve cells expressing hamster PrP. The presence of endogenous mouse PrP in hamster PrP-expressing CAD5 cells completely blocked the replication of hamster prions. Interestingly, murine N2a neuroblastoma cells ablated for endogenous PrP expression were susceptible to mouse prions, but not hamster prions upon expression of cognate PrP.

**Conclusions:** Our results demonstrate that CAD5 cells, but not N2a cells, can propagate prion strains from non-mouse species following the ablation of endogenous mouse PrP. This suggests that CAD5 cells either possess cellular factors that enhance or lack factors that restrict the diversity of strains that can be propagated. We conclude that transfected CAD5-PrP<sup>-/-</sup> cells may be a useful tool for assessing the biology of prion strains and dissecting the mechanism of prion replication.

## References

[1] Mahal et al., Proc Natl Acad Sci USA. 2007;104 (52):20,908-13.

[2] Berry et al., Proc Natl Acad Sci USA. 2013;110(44): E4160-9.

## 170. Biochemical characterization of PrPSc species at pre-clinical stages of disease in a mouse model of prion disease (RML)

Ghazaleh Eskandari-Sedighi<sup>a,b</sup>, Leonardo Cortez<sup>a,c</sup>, Jing Yang<sup>a</sup>, Hristina Gapeshina<sup>a</sup>, Nathalie Daude<sup>a</sup>, Valerie Sim<sup>a,c</sup> and David Westaway<sup>a,b,d</sup>

<sup>a</sup>Centre for prions and protein folding diseases, University of Alberta, Edmonton, Alberta, Canada; <sup>b</sup>Department of Biochemistry, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada; <sup>c</sup>Department of Medicine, Division of Neurology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada; <sup>d</sup>Department of Medicine, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada

CONTACT Ghazaleh Eskandari-Sedighi ghazaleh@ualberta.ca

## ABSTRACT

**Background:** It is known that the amount of PrPSc and infectious titre in brains of infected animals can plateau at sub-clinical stages, suggesting a division of prion infectivity and toxicity phases [1]. Previous work in our lab has shown that a post-translational downregulation of PrPC could account the plateau effect [2,3]. These data also suggested that diminishing levels of PrPC (i.e. the substrate for PrPSc formation), as well as the rise in accumulated PrPSc are important determinants in the transition from sub-clinical to clinical stage. Although transcriptomic studies point at major role of inflammation in prion diseases [4], and a recent study on global metabolomics in mice points at changes in brain chemistry at a mid-stage in disease progression [5], there is still ambiguity about the causal relationship between these changes, neuronal loss and clinical manifestation of the disease.

**Material and Methods:** Using mice inoculated with the RML isolate of mouse-adapted scrape, we applied different techniques to track the aforementioned parameters and the biochemical features of the PrPSc particles. The isotropic fractionator technique [6] was used to quantify the number of neural and non-neural cells in the brain of infected animals. Asymmetric-flow field- flow fractionation (AF4), a one-phase chromatography technique that separates particles based on their size, was used to isolate the PrPSc particles from brains; size of the isolated particles was determined by dynamic light scattering and the seeding activity was evaluated by RT-QuIC. Histopathology was used to monitor spongiosis changes, PrPSc accumulation in the brain and astrocytes activation.

**Results:** Spongiosis, PrPSc accumulation in the brain and astrocyte activation were detected in brains from
<figure><img src="image_107.png" /></figure>

<sup>a</sup>Tanz Centre for Research in Neurodegenerative Diseases, Toronto, Canada; <sup>b</sup>Department of Biochemistry, University of Toronto, Toronto, Canada; <sup>c</sup>Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada 4 Department of Medical Microbiology and Immunology, Creighton University, Omaha, USA

CONTACT Matthew Bourkas matthew.bourkas@mail.utoronto.ca

## ABSTRACT

**Background:** Prion research has been hindered by a lack of cellular paradigms for studying the replication of prions from different species. Although hamster prions have been widely used to study prion replication in animals and within *in vitro* amplification systems, they have proven challenging to propagate in cultured cells. Since the murine catecholaminergic cell line CAD5 is uniquely susceptible to a diverse range of mouse prion strains, [1,2] we hypothesized that it might also be capable of propagating non-mouse prions.

**Materials and Methods:** We generated CAD5 cells lacking endogenous $PrP^C$ (CAD5-$PrP^{−/−}$ cells) using CRISPR/Cas9-mediated genome editing. CAD5-$PrP^{−/−}$ cells were stably transfected with a plasmid encoding hamster PrP and then challenged with brain homogenates containing various strains of hamster prions.

**Results:** When exposed to the 263K, HY, or 139H strains, hamster PrP-expressing CAD5-$PrP^{−/−}$ cells stably propagated high levels of protease-resistant PrP. Moreover, when these cells were challenged with 10-fold serial dilutions of 263K prions, PK-resistant PrP was obtained with dilutions up to and including 0.000002% brain homogenate. Cellular homogenates from 263K-infected cells exhibited prion seeding activity in the RT-QuIC assay and were infectious to naïve cells expressing hamster PrP. The presence of endogenous mouse PrP in hamster PrP-expressing CAD5 cells completely blocked the replication of hamster prions. Interestingly, murine N2a neuroblastoma cells ablated for endogenous PrP expression were susceptible to mouse prions, but not hamster prions upon expression of cognate PrP.

**Conclusions:** Our results demonstrate that CAD5 cells, but not N2a cells, can propagate prion strains from non-mouse species following the ablation of endogenous mouse PrP. This suggests that CAD5 cells either possess cellular factors that enhance or lack factors that restrict the diversity of strains that can be propagated. We conclude that transfected CAD5-$PrP^{−/−}$ cells may be a useful tool for assessing the biology of prion strains and dissecting the mechanism of prion replication.

## References

[1] Mahal et al., Proc Natl Acad Sci USA. 2007;104 (52):20,908-13.

[2] Berry et al., Proc Natl Acad Sci USA. 2013;110(44): E4160-9.

## 170. Biochemical characterization of PrPSc species at pre-clinical stages of disease in a mouse model of prion disease (RML)

Ghazaleh Eskandari-Sedighi<sup>a,b</sup>, Leonardo Cortez<sup>a,c</sup>, Jing Yang<sup>a</sup>, Hristina Gapeshina<sup>a</sup>, Nathalie Daude<sup>a</sup>, Valerie Sim<sup>a,c</sup> and David Westaway<sup>a,b,d</sup>

<sup>a</sup>Centre for prions and protein folding diseases, University of Alberta, Edmonton, Alberta, Canada; <sup>b</sup>Department of Biochemistry, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada; <sup>c</sup>Department of Medicine, Division of Neurology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada; <sup>d</sup>Department of Medicine, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada

CONTACT Ghazaleh Eskandari-Sedighi ghazaleh@ualberta.ca

## ABSTRACT

**Background:** It is known that the amount of PrPSc and infectious titre in brains of infected animals can plateau at sub-clinical stages, suggesting a division of prion infectivity and toxicity phases [1]. Previous work in our lab has shown that a post-translational downregulation of PrPC could account the plateau effect [2,3]. These data also suggested that diminishing levels of PrPC (i.e. the substrate for PrPSc formation), as well as the rise in accumulated PrPSc are important determinants in the transition from sub-clinical to clinical stage. Although transcriptomic studies point at major role of inflammation in prion diseases [4], and a recent study on global metabolomics in mice points at changes in brain chemistry at a mid-stage in disease progression [5], there is still ambiguity about the causal relationship between these changes, neuronal loss and clinical manifestation of the disease.

**Material and Methods:** Using mice inoculated with the RML isolate of mouse-adapted scrape, we applied different techniques to track the aforementioned parameters and the biochemical features of the PrPSc particles. The isotropic fractionator technique [6] was used to quantify the number of neural and non-neural cells in the brain of infected animals. Asymmetric-flow field-flow fractionation (AF4), a one-phase chromatography technique that separates particles based on their size, was used to isolate the PrPSc particles from brains; size of the isolated particles was determined by dynamic light scattering and the seeding activity was evaluated by RT-QuIC. Histopathology was used to monitor spongiosis changes, PrPSc accumulation in the brain and astrocytes activation.

**Results:** Spongiosis, PrPSc accumulation in the brain and astrocyte activation were detected in brains from
<figure><img src="image_108.png" /></figure>

90 dpi. However, significant neuronal loss was only validated at the terminal stage of the disease, ca. 150 dpi. Biochemical characterization of brain-derived PrPSc species at different timepoints is currently in progress.

**Conclusions:** Our quantitative data measuring NeuN positive nuclei concur with previous histological assessments in indicating that neuronal loss is a late event in prion diseases and thus cannot account for preclinical reductions in steady-state levels of PrPC. Yet earlier chemical changes can be detected in brain as early as 90 dpi. We aim to pinpoint the early PrPSc transition events by the FFF technique and understand the causal events that trigger clinical symptoms of the disease.

## References

[1] Sandberg et al., Nature 2011;470:540–542.

[2] Mays et al., J Clin Invest. 2014;24(2):847–858.

[3] Mays et al., J Virology. 2015;89(24):12,418–1242.

[4] Vincenti et al., J Virology. 2016;90(6):3003–3017.

[5] Bourgognon et al., Cell Death and Differentiation. 2018;25:1408–1425.

[6] Herculano-Houzel, J Neurosci. 2005;5(10):2518–2521.

# 171. Efficacy of short-term exposure to sodium hypochlorite (20,000 ppm) or 4% acetic acid sodium lauryl sulfate (4% acetic SDS) to inactivate chronic wasting disease infectivity in a laboratory setting

Erin E McNulty, Amy V Nalls, Laura A Pulscher, Candace K Mathiason

Colorado State University, Fort Collins, Colorado, USA

CONTACT Erin E McNulty eem@colostate.edu

## ABSTRACT

Prions are highly resistant to inactivation by conventional sterilization processes and disinfecting agents. The Center for Disease Control (CDC) recommendation for decontamination of the infectious agent associated with prions include exposure to 20,000 parts per million (ppm) sodium hypochlorite for 30–60 min, or high pressure-autoclave at 134 C for 1 h. High pressure-high temperature autoclaving is typically used to inactivate prions present on plastics, personal protective equipment and within small quantity tissues/homogenates routinely used by laboratory personnel. Laboratory benches, equipment and instruments are routinely exposed to 20,000 ppm sodium hypochlorite generated by diluting household bleach 1:2.5. Due to the corrosive nature of high sodium hypochlorite

concentrations investigators have searched for effective means to inactivate prions that will result in less damage to the laboratory setting. To this end 4% acetic acid sodium lauryl sulfate (4% acetic SDS) has been incorporated into prion laboratory cleaning regimens. In this study we explore the efficacy of short-term exposure to 20,000 ppm hypochlorite or 4% acetic SDS to inactivate prion infectivity in a laboratory setting by use of amplification and bio-assays.

To emulate a more typical laboratory scenario, a film of brain homogenate (10% w/v) from naïve or chronic wasting disease (CWD)-infected deer was applied to a laboratory bench free of prion contamination. Each film was treated by mist of water, 20,000 ppm hypochlorite or 4% acetic acid and left for 30–60 s prior to cleanup. A swab was used to swipe the area post cleanup. Fluid retrieved from the swab was assessed for infectivity in TgCer5037 mouse bioassay.

We found that 0/9 mice inoculated with 20,000 ppm hypochlorite-treated and 5/6 mice inoculated with 4% acetic SDS-treated CWD inoculum demonstrated clinical disease consistent with prion infection. Development of terminal clinical disease in mice inoculated with 4% acetic SDS was prolonged by 50% as compared to no treatment controls. PrP<sup>Sc</sup> deposition and amyloid seeding activity was demonstrated by immunohistochemistry and PMCA in all mice showing clinical disease. Mice inoculated with similarly treated naïve brain homogenate remained free of prion disease.

A contemporary dilutional bioassay permitted us to estimate the infectivity remaining in mice inoculated with treated inoculum that succumbed to prion infection. We demonstrate that a laboratory cleaning regimen including short-term exposure to 20,000 ppm hypochlorite results in sterilizing inactivation of CWD prion infectivity while 4% acetic SDS decreased CWD burden by 17%. This study provides findings to guide development of prion laboratory cleaning procedures.

# 172. Establishment of PrP<sup>CWD</sup> extraction and detection methods in the farm soil

Kyung Je Park, Hoo Chang Park, In Soon Roh, Hyo Jin Kim, Hae-Eun Kang and Hyun Joo Sohn

Foreign Animal Disease Division, Animal and Plant Quarantine Agency, Gimcheon, Gyeongsangbuk-do, Korea

## ABSTRACT

**Introduction:** Transmissible spongiform encephalopathy (TSE) is a fatal neurodegenerative disorder, which is so-called as prion diseases due to the causative agents (PrP<sup>Sc</sup>). TSEs are believed to be due to the template-
<figure><img src="image_108.png" /></figure>

90 dpi. However, significant neuronal loss was only validated at the terminal stage of the disease, ca. 150 dpi. Biochemical characterization of brain-derived PrPSc species at different timepoints is currently in progress.

**Conclusions:** Our quantitative data measuring NeuN positive nuclei concur with previous histological assessments in indicating that neuronal loss is a late event in prion diseases and thus cannot account for preclinical reductions in steady-state levels of PrPC. Yet earlier chemical changes can be detected in brain as early as 90 dpi. We aim to pinpoint the early PrPSc transition events by the FFF technique and understand the causal events that trigger clinical symptoms of the disease.

## References

[1] Sandberg et al., Nature 2011;470:540–542.

[2] Mays et al., J Clin Invest. 2014;24(2):847–858.

[3] Mays et al., J Virology. 2015;89(24):12,418–1242.

[4] Vincenti et al., J Virology. 2016;90(6):3003–3017.

[5] Bourgognon et al., Cell Death and Differentiation. 2018;25:1408–1425.

[6] Herculano-Houzel, J Neurosci. 2005;5(10):2518–2521.

# 171. Efficacy of short-term exposure to sodium hypochlorite (20,000 ppm) or 4% acetic acid sodium lauryl sulfate (4% acetic SDS) to inactivate chronic wasting disease infectivity in a laboratory setting

Erin E McNulty, Amy V Nalls, Laura A Pulscher, Candace K Mathiason

Colorado State University, Fort Collins, Colorado, USA

CONTACT Erin E McNulty eem@colostate.edu

## ABSTRACT

Prions are highly resistant to inactivation by conventional sterilization processes and disinfecting agents. The Center for Disease Control (CDC) recommendation for decontamination of the infectious agent associated with prions include exposure to 20,000 parts per million (ppm) sodium hypochlorite for 30–60 min, or high pressure-autoclave at 134 C for 1 h. High pressure-high temperature autoclaving is typically used to inactivate prions present on plastics, personal protective equipment and within small quantity tissues/homogenates routinely used by laboratory personnel. Laboratory benches, equipment and instruments are routinely exposed to 20,000 ppm sodium hypochlorite generated by diluting household bleach 1:2.5. Due to the corrosive nature of high sodium hypochlorite

concentrations investigators have searched for effective means to inactivate prions that will result in less damage to the laboratory setting. To this end 4% acetic acid sodium lauryl sulfate (4% acetic SDS) has been incorporated into prion laboratory cleaning regimens. In this study we explore the efficacy of short-term exposure to 20,000 ppm hypochlorite or 4% acetic SDS to inactivate prion infectivity in a laboratory setting by use of amplification and bio-assays.

To emulate a more typical laboratory scenario, a film of brain homogenate (10% w/v) from naïve or chronic wasting disease (CWD)-infected deer was applied to a laboratory bench free of prion contamination. Each film was treated by mist of water, 20,000 ppm hypochlorite or 4% acetic acid and left for 30–60 s prior to cleanup. A swab was used to swipe the area post cleanup. Fluid retrieved from the swab was assessed for infectivity in TgCer5037 mouse bioassay.

We found that 0/9 mice inoculated with 20,000 ppm hypochlorite-treated and 5/6 mice inoculated with 4% acetic SDS-treated CWD inoculum demonstrated clinical disease consistent with prion infection. Development of terminal clinical disease in mice inoculated with 4% acetic SDS was prolonged by 50% as compared to no treatment controls. PrP<sup>Sc</sup> deposition and amyloid seeding activity was demonstrated by immunohistochemistry and PMCA in all mice showing clinical disease. Mice inoculated with similarly treated naïve brain homogenate remained free of prion disease.

A contemporary dilutional bioassay permitted us to estimate the infectivity remaining in mice inoculated with treated inoculum that succumbed to prion infection. We demonstrate that a laboratory cleaning regimen including short-term exposure to 20,000 ppm hypochlorite results in sterilizing inactivation of CWD prion infectivity while 4% acetic SDS decreased CWD burden by 17%. This study provides findings to guide development of prion laboratory cleaning procedures.

# 172. Establishment of PrP<sup>CWD</sup> extraction and detection methods in the farm soil

Kyung Je Park, Hoo Chang Park, In Soon Roh, Hyo Jin Kim, Hae-Eun Kang and Hyun Joo Sohn

Foreign Animal Disease Division, Animal and Plant Quarantine Agency, Gimcheon, Gyeongsangbuk-do, Korea

## ABSTRACT

**Introduction:** Transmissible spongiform encephalopathy (TSE) is a fatal neurodegenerative disorder, which is so-called as prion diseases due to the causative agents (PrP<sup>Sc</sup>). TSEs are believed to be due to the template-
<figure><img src="image_109.png" /></figure>

directed accumulation of disease-associated prion protein, generally designated $PrP^{Sc}$. Chronic wasting disease (CWD) is the prion disease that is known spread horizontally. CWD has confirmed last in Republic of Korea in 2016 since first outbreak of CWD in 2001. The environmental reservoirs mediate the transmission of this disease. The significant levels of infectivity have been detected in the saliva, urine, and faeces of TSE-infected animals. Soil can serve as a stable reservoir for infectious prion proteins. We found that $PrP^{CWD}$ can be extracted and detected in CWD contaminated soil which has kept at room temperature until 4 years after 0.001 ~ 1% CWD exposure and natural CWD-affected farm soil through PBS washing and sPMCAb.

**Materials and Methods: Procedure of serial PMCAb.** CWD contaminated soil which has kept at room temperature (RT) for 1 ~ 4 year after 0.001% ~1% CWD brain homogenates exposure for 4 months collected 0.14 g. The soil was collected by the same method once of year until 4 year after stop CWD exposure. We had conducted the two steps. There are two kinds of 10 times washing step and one amplification step. The washing step was detached $PrP^{Sc}$ from contaminated soil by strong vortex with maximum rpm. We harvest supernatant every time by 10 times. As the other washing step, the Washed soil was made by washing 10 times soil using slow rotator and then harvest resuspended PBS for removing large impurity material. Last step was prion amplification step for detection of $PrP^{CWD}$ in soil supernatant and the washed soil by sPMCAb. Normal brain homogenate (NBH) was prepared by homogenization of brains with glass dounce in 9 volumes of cold PBS with TritonX-100, 5 mM EDTA, 150 mM NaCl and 0.05% Digitonin (sigma) plus Complete mini protease inhibitors (Roche) to a final concentration of 5%(w/v) NBHs were centrifuged at 2000 g for 1 min, and supernatant removed and frozen at -70 C for use. CWD consisted of brain from natural case in Korea and was prepared as 10%(w/v) homogenate. Positive sample was diluted to a final dilution 1:1000 in NBH, with serial 3:7 dilutions in NBH. Sonication was performed with a Misonix 4000 sonicator with amplitude set to level 70, generating an average output of 160W with two teflon beads during each cycle. One round consisted of 56 cycles of 30 s of sonication followed 9 min 30 s of 37°C incubation. **Western Blotting (WB) for $PrP^{Sc}$ detection.** The samples (20 µL) after each round of amplification were mixed with proteinase K (2 mg/ml) and incubated 37°C for 1 h. Samples were separated by SDS-PAGE

and transferred onto PVDF membrane. After blocking, the membrane was incubated for 1 h with 1st antibody S1 anti rabbit serum (APQA, 1:3000) and developed with enhanced chemiluminescence detection system.

**Results:** We excluded from first to third supernatant in view of sample contamination. It was confirmed abnormal $PrP$ amplification in all soil supernatants from fourth to tenth. From 0.01% to 1% contaminated washed soils were identified as abnormal prions. 0.001% contaminated washed soil did not show $PrP$ specific band (Fig 1). The soil was collected by the same method once of year until 4 year after stop CWD exposure. After sPMCAb there were no $PrP^{CWD}$ band in from second to fourth year 0.001% washed soil. but It was confirmed that the abnormal prion was amplified in the washing supernatant which was not amplified in the washed soil. we have decided to use soil supernatant for soil testing (Fig. 2). After third rounds of amplification, $PrP^{Sc}$ signals observed in three out of four sites from CWD positive farm playground. No signals were observed in all soil samples from four CWD negative farm (Fig. 3).

**Conclusions:** Our studies showed that $PrP^{CWD}$ persist in 0.001% CWD contaminated soil for at least 4 year and natural CWD-affected farm soil. When cervid reintroduced into CWD outbreak farm, the strict decontamination procedures of the infectious agent should be performed in the environment of CWD-affected cervid habitat.

**References**

[1] Nagooka K et al., Sensitive detection of scrapie prion protein in soil. Biochem Biophys Res Commun. 2010;397:626-630.

[2] Georgsson G et al., Infectious agent of sheep scrapie may persist in the environment for at least 16 years. J of Gen Virol. 2006;87:3737-3740.

[3] Saunders SE et al., Prions adhere to soil minerals and remain infectious. Plos Pathog. 2006;2:296-302.

[4] Tamguney G et al., Asymptomatic deer excerete infectious prions faces. Nature. 2009;529-532.

**173. Antisense oligonucleotides for the treatment of neurodegenerative diseases**

Hien T Zhao, Holly Kordasiewicz, Dan Norris, Anne Smith, Roger Lane, C. Frank Bennett and Eric Swayze

Ionis Pharmaceuticals, Inc. Carlsbad, CA,USA

CONTACT Hien T Zhao hzhao@ionisph.com
<figure><img src="image_109.png" /></figure>

directed accumulation of disease-associated prion protein, generally designated $PrP^{Sc}$. Chronic wasting disease (CWD) is the prion disease that is known spread horizontally. CWD has confirmed last in Republic of Korea in 2016 since first outbreak of CWD in 2001. The environmental reservoirs mediate the transmission of this disease. The significant levels of infectivity have been detected in the saliva, urine, and faeces of TSE-infected animals. Soil can serve as a stable reservoir for infectious prion proteins. We found that $PrP^{CWD}$ can be extracted and detected in CWD contaminated soil which has kept at room temperature until 4 years after 0.001 ~ 1% CWD exposure and natural CWD-affected farm soil through PBS washing and sPMCAb.

**Materials and Methods: Procedure of serial PMCAb.** CWD contaminated soil which has kept at room temperature (RT) for 1 ~ 4 year after 0.001% ~1% CWD brain homogenates exposure for 4 months collected 0.14 g. The soil was collected by the same method once of year until 4 year after stop CWD exposure. We had conducted the two steps. There are two kinds of 10 times washing step and one amplification step. The washing step was detached $PrP^{Sc}$ from contaminated soil by strong vortex with maximum rpm. We harvest supernatant every time by 10 times. As the other washing step, the Washed soil was made by washing 10 times soil using slow rotator and then harvest resuspended PBS for removing large impurity material. Last step was prion amplification step for detection of $PrP^{CWD}$ in soil supernatant and the washed soil by sPMCAb. Normal brain homogenate (NBH) was prepared by homogenization of brains with glass dounce in 9 volumes of cold PBS with TritonX-100, 5 mM EDTA, 150 mM NaCl and 0.05% Digitonin (sigma) plus Complete mini protease inhibitors (Roche) to a final concentration of 5%(w/v) NBHs were centrifuged at 2000 g for 1 min, and supernatant removed and frozen at -70 C for use. CWD consisted of brain from natural case in Korea and was prepared as 10%(w/v) homogenate. Positive sample was diluted to a final dilution 1:1000 in NBH, with serial 3:7 dilutions in NBH. Sonication was performed with a Misonix 4000 sonicator with amplitude set to level 70, generating an average output of 160W with two teflon beads during each cycle. One round consisted of 56 cycles of 30 s of sonication followed 9 min 30 s of 37°C incubation. **Western Blotting (WB) for $PrP^{Sc}$ detection.** The samples (20 µL) after each round of amplification were mixed with proteinase K (2 mg/ml) and incubated 37°C for 1 h. Samples were separated by SDS-PAGE

and transferred onto PVDF membrane. After blocking, the membrane was incubated for 1 h with 1st antibody S1 anti rabbit serum (APQA, 1:3000) and developed with enhanced chemiluminescence detection system.

**Results:** We excluded from first to third supernatant in view of sample contamination. It was confirmed abnormal $PrP$ amplification in all soil supernatants from fourth to tenth. From 0.01% to 1% contaminated washed soils were identified as abnormal prions. 0.001% contaminated washed soil did not show $PrP$ specific band (Fig 1). The soil was collected by the same method once of year until 4 year after stop CWD exposure. After sPMCAb there were no $PrP^{CWD}$ band in from second to fourth year 0.001% washed soil. but It was confirmed that the abnormal prion was amplified in the washing supernatant which was not amplified in the washed soil. we have decided to use soil supernatant for soil testing (Fig. 2). After third rounds of amplification, $PrP^{Sc}$ signals observed in three out of four sites from CWD positive farm playground. No signals were observed in all soil samples from four CWD negative farm (Fig. 3).

**Conclusions:** Our studies showed that $PrP^{CWD}$ persist in 0.001% CWD contaminated soil for at least 4 year and natural CWD-affected farm soil. When cervid reintroduced into CWD outbreak farm, the strict decontamination procedures of the infectious agent should be performed in the environment of CWD-affected cervid habitat.

**References**

[1] Nagooka K et al., Sensitive detection of scrapie prion protein in soil. Biochem Biophys Res Commun. 2010;397:626-630.

[2] Georgsson G et al., Infectious agent of sheep scrapie may persist in the environment for at least 16 years. J of Gen Virol. 2006;87:3737-3740.

[3] Saunders SE et al., Prions adhere to soil minerals and remain infectious. Plos Pathog. 2006;2:296-302.

[4] Tamguney G et al., Asymptomatic deer excerete infectious prions faces. Nature. 2009;529-532.

**173. Antisense oligonucleotides for the treatment of neurodegenerative diseases**

Hien T Zhao, Holly Kordasiewicz, Dan Norris, Anne Smith, Roger Lane, C. Frank Bennett and Eric Swayze

Ionis Pharmaceuticals, Inc. Carlsbad, CA,USA

CONTACT Hien T Zhao hzhao@ionisph.com
<figure><img src="image_110.png" /></figure>

## ABSTRACT

Antisense oligonucleotides (ASOs) are emerging as a viable therapeutic approach to alter production of proteins implicated in currently untreatable neurodegenerative diseases. ASOs are single stranded nucleotides, typically 20 bases in length, that bind complementary target RNA through Watson and Crick hybridization. Depending on ASO design, hybridization can lead to selective degradation of the target RNA, alteration of RNA splicing or another highly specific post-transcriptional modification. Foundational science tools are available to facilitate transition from the bench to the clinic, as evidenced by regulatory approval of an ASO for the treatment of spinal muscular atrophy and initiation of clinical trials for ASOs in Huntington's disease, amyotrophic lateral sclerosis and other neurological conditions. Tools that guide drug development and mitigate risk include pharmacology studies in rodents to demonstrate proof-of-concept phenotypic benefit of ASOs and biodistribution studies in larger species to characterize pharmacokinetics and pharmacodynamics of the ASO and key target-engagement markers. Additionally, PKPD models can be built using preclinical data to predict ASO effects in humans, enabling informed selection of ASO doses for clinical trials and interpretation of data collected in the clinic. Here, we describe our experiences in ASO development for neurodegenerative diseases, including proof-of-concept and biodistribution animal studies, PKPD model-building and clinical trial design and interpretation. These experiences provide a blueprint for advancement of ASO therapies designed to treat neurodegenerative diseases, including prion disease.

# 174. Developing a gene reporter system allowing real-time in-vivo monitoring of prion disease progression

Matthew J. Martinª and Stephanie A. Boothª,ᵇ

CONTACT Matthew J. Martin Matthew.martin4@canada.ca

## ABSTRACT

**Introduction:** Prion diseases are a fatal neurodegenerative disease caused by the misfolding of the cellular prion protein, PrPC into an infectious isoform, PrPSc. Accumulation of this infectious isoform leads to the damage and death of neurons, and the progression of the

disease. The molecular events involved in the pathogenesis of this disease are poorly understood and uncovering them has proven challenging due to the difficulties of identifying and isolating degenerating neurons for analysis. Ultimately, the aim of this project is to engineer adeno-associated viruses (AAV) to deliver genetic tools into neurons to drive the expression of reporter molecules during neurodegeneration. This would enable real-time monitoring of the disease *in vivo*, and identification of specific neurons to be isolated for further analysis.

**Materials and Methods:** AAV rh10 was used to deliver a genetic construct containing a short hairpin RNA sequence targeting the cellular prion gene to test the expression system. This virus construct was injected into newborn mice via their superficial temporal vein. This short hairpin RNA is designed to post-transcriptionally silence prion expression, inducing immunity against prions to the transduced cell. The genetic construct also expresses a red fluorescent protein to be used as a biomarker signalling transduced cells. At 7 weeks of age, the mice were infected with Rocky Mountain Laboratory prions, or mock-infected as a control. Brain, liver, lungs, spleen, and serum samples was collected at 60, 90, and 141 days post-infection. IHC is used to determine the presence of the virus in these tissues and Laser Capture Microdissection used to specifically isolate transduced cells for further analysis of their RNA and protein content.

**Results:** The distribution of the AAV rh10 throughout multiple regions of mouse brain will be described as well as the relative transduction of neurons versus microglia and astrocytes. In addition, the kinetics of the vector concentration in the brain over time will be described. This work is the basis for engineering vectors capable of targeting specific neurons in mouse brain tissue in future studies to understand prion pathogenesis and prion strains *in vivo*.

# 175. Environmental pathways of CWD prions

Alsu Kuznetsovaª, Debbie McKenzieᵇ and Judd M. Aikenª

CONTACT Alsu Kuznetsov alsu@ualberta.ca

## ABSTRACT

Soil can serve as a reservoir and route for horizontal transmission of chronic wasting disease (CWD) by interaction with the infectious prion protein

<footnote>ªUniversity of Manitoba; ᵇNational Microbiology Laboratory, Public Health Agency of Canada</footnote>

<footnote>ªAgricultural Life and Environmental Sciences Faculty, University of Alberta, Edmonton, Canada; ᵇFaculty of Science, University of Alberta, Edmonton, Canada</footnote>
<figure><img src="image_110.png" /></figure>

## ABSTRACT

Antisense oligonucleotides (ASOs) are emerging as a viable therapeutic approach to alter production of proteins implicated in currently untreatable neurodegenerative diseases. ASOs are single stranded nucleotides, typically 20 bases in length, that bind complementary target RNA through Watson and Crick hybridization. Depending on ASO design, hybridization can lead to selective degradation of the target RNA, alteration of RNA splicing or another highly specific post-transcriptional modification. Foundational science tools are available to facilitate transition from the bench to the clinic, as evidenced by regulatory approval of an ASO for the treatment of spinal muscular atrophy and initiation of clinical trials for ASOs in Huntington's disease, amyotrophic lateral sclerosis and other neurological conditions. Tools that guide drug development and mitigate risk include pharmacology studies in rodents to demonstrate proof-of-concept phenotypic benefit of ASOs and biodistribution studies in larger species to characterize pharmacokinetics and pharmacodynamics of the ASO and key target-engagement markers. Additionally, PKPD models can be built using preclinical data to predict ASO effects in humans, enabling informed selection of ASO doses for clinical trials and interpretation of data collected in the clinic. Here, we describe our experiences in ASO development for neurodegenerative diseases, including proof-of-concept and biodistribution animal studies, PKPD model-building and clinical trial design and interpretation. These experiences provide a blueprint for advancement of ASO therapies designed to treat neurodegenerative diseases, including prion disease.

# 174. Developing a gene reporter system allowing real-time in-vivo monitoring of prion disease progression

Matthew J. Martinª and Stephanie A. Boothª,ᵇ

CONTACT Matthew J. Martin Matthew.martin4@canada.ca

## ABSTRACT

**Introduction:** Prion diseases are a fatal neurodegenerative disease caused by the misfolding of the cellular prion protein, PrPC into an infectious isoform, PrPSc. Accumulation of this infectious isoform leads to the damage and death of neurons, and the progression of the

disease. The molecular events involved in the pathogenesis of this disease are poorly understood and uncovering them has proven challenging due to the difficulties of identifying and isolating degenerating neurons for analysis. Ultimately, the aim of this project is to engineer adeno-associated viruses (AAV) to deliver genetic tools into neurons to drive the expression of reporter molecules during neurodegeneration. This would enable real-time monitoring of the disease *in vivo*, and identification of specific neurons to be isolated for further analysis.

**Materials and Methods:** AAV rh10 was used to deliver a genetic construct containing a short hairpin RNA sequence targeting the cellular prion gene to test the expression system. This virus construct was injected into newborn mice via their superficial temporal vein. This short hairpin RNA is designed to post-transcriptionally silence prion expression, inducing immunity against prions to the transduced cell. The genetic construct also expresses a red fluorescent protein to be used as a biomarker signalling transduced cells. At 7 weeks of age, the mice were infected with Rocky Mountain Laboratory prions, or mock-infected as a control. Brain, liver, lungs, spleen, and serum samples was collected at 60, 90, and 141 days post-infection. IHC is used to determine the presence of the virus in these tissues and Laser Capture Microdissection used to specifically isolate transduced cells for further analysis of their RNA and protein content.

**Results:** The distribution of the AAV rh10 throughout multiple regions of mouse brain will be described as well as the relative transduction of neurons versus microglia and astrocytes. In addition, the kinetics of the vector concentration in the brain over time will be described. This work is the basis for engineering vectors capable of targeting specific neurons in mouse brain tissue in future studies to understand prion pathogenesis and prion strains *in vivo*.

# 175. Environmental pathways of CWD prions

Alsu Kuznetsovaª, Debbie McKenzieᵇ and Judd M. Aikenª

CONTACT Alsu Kuznetsov alsu@ualberta.ca

## ABSTRACT

Soil can serve as a reservoir and route for horizontal transmission of chronic wasting disease (CWD) by interaction with the infectious prion protein

<footnote>ªUniversity of Manitoba; ᵇNational Microbiology Laboratory, Public Health Agency of Canada</footnote>

<footnote>ªAgricultural Life and Environmental Sciences Faculty, University of Alberta, Edmonton, Canada; ᵇFaculty of Science, University of Alberta, Edmonton, Canada</footnote>