<figure><img src="image_72.png" /></figure>

variation regarding prion protein glycosylation. The analysis was performed on VRQ/VRQ sheep brain affected with classical scrapie. The ovine PrP<sup>Sc</sup> was isolated and purified by immunopurification and SDS-PAGE. The isolation of the scrapie prion protein was shown to be somewhat challenging, since glycoproteins are heterogeneously glycosylated, this leads to a decreased concentration of each analyte (each specific glycan). After obtaining a sufficient amount and purifying the protein, the di- and monoglycosylated forms of the prion protein were extracted from the gel and in-gel trypsin digestion was performed. The tryptic glycopeptides were then analysed using reverse phase liquid chromatography coupled with electrospray mass spectrometry (LC-ESI-MS). The glycan structures found on the ovine PrP<sup>Sc</sup> have been shown to contain 'brain-specific' N-glycans, which are high in fucose and sialic acids. Other features found, that are characteristic for brain N-glycans, include hybrid-type structures, bisecting N-Acetylglucosamine (GlcNAc) and core (α1-6)fucose, as well as outer arm (α1-3)fucose. We were able to develop a method for isolation of ovine PrP<sup>Sc</sup> from infected tissue and analysing the glycopeptides with LC-ESI-MS. With this approach we plan to compare glycan structures between different ovine prion strains in order to better understand whether or not N-glycans contribute to prion strain differences.

**KEYWORDS:** Prion; N-glycan; LC-ESI-MS

**110. Involvement of MAPK pathway and tau in pα-syn*-induced mitotoxicity**

Diego Grassi<sup>a,b</sup>, Natalia Diaz-Perez<sup>c</sup>, Laura A. Volpicelli-Daley<sup>d</sup> and Corinne Ida Lasmézas<sup>a,b</sup>

**CONTACT** Corinne Ida Lasmézas lasmezas@scripps.edu

**ABSTRACT**

We recently identified a phosphorylated form of α-synuclein, pα-syn*, as a key neurotoxic α-synuclein species found in cultured neurons, as well as in mouse and Parkinson's disease patients' brains. Small pα-syn* aggregates localize to mitochondria and induce mitochondrial damage and fragmentation. Herein, we investigated the molecular basis of pα-syn*-induced toxicity. By immunofluorescence, we found phosphorylated MKK4, JNK, ERK5 and p38 MAPKs in pα-syn* inclusions. pJNK colorized with pα-syn* at mitochondria and mitochondria-

associated ER membranes where it was associated with BiP and pACC1. We also found that pα-syn* aggregates are tightly associated with small ptau aggregates of similar size. Pα-syn*/ptau inclusions localized to areas of mitochondrial damage and to mitophagic vesicles, showing their role in mitochondrial toxicity, mitophagy induction, and their removal along with damaged mitochondrial fragments. Several MAPKs may act cooperatively to phosphorylate tau, notably JNK, p38, and GSK3β, a non-MAPK that was also found phosphorylated in the vicinity of pα-syn*/ptau aggregates. Thus, pα-syn* appears to be the trigger of a series of kinase mediated pathogenic events and a link between α-syn pathology and tau, another protein known to aggregate in Parkinson's disease and other synucleinopathies.

**KEYWORDS:** Alpha-synuclein; tau; Parkinson's disease; primary neurons; kinases; neurotoxicity; mitochondria; mitophagy

**111. Identification of rare genetic variants associated with sporadic CJD by exome sequencing**

Helen Speedy<sup>a</sup>, Penny Norsworthy<sup>a</sup>, Holger Hummerich<sup>a</sup>, Gary Adamson<sup>a</sup>, James Uphill<sup>a</sup>, John Collinge<sup>a</sup> and Simon Mead<sup>a</sup>

**CONTACT** Helen Speedy h.speedy@ucl.ac.uk

**ABSTRACT**

**Background:** Prion diseases are progressive neurodegenerative conditions characterized by the misfolding and aggregation of prion protein. Sporadic Creutzfeldt-Jakob Disease (sCJD) is the most common human prion disease. The common polymorphism at codon 129 of the *PRNP* gene influences sCJD risk; however additional genetic factors are thought to contribute to disease susceptibility. Ongoing genome-wide association studies have identified common genetic variants associated with sCJD risk. In this study we use exome sequencing to investigate the role of rare, disruptive variants as determinants of susceptibility to sCJD.

**Materials and Methods:** This study incorporates exome sequencing of an in-house set of 249 sCJD cases as well as data from 126 sCJD cases from the Medical Research Council (MRC) Brain Bank Network. Controls are derived from publicly available resources.

In-house, genomic DNA was extracted from peripheral blood. Exome capture using Agilent technology and paired-end sequencing on an Illumina HiSeq2000 sequencer was performed at Source Bioscience (Nottingham, UK).

<footnote><sup>a</sup>Department of Immunology and Microbiology, The Scripps Research Institute, Scripps Florida, Jupiter, FL, USA; <sup>b</sup>Department of Neuroscience, The Scripps Research Institute, Scripps Florida, Jupiter, FL, USA; <sup>c</sup>Private address, Palm Beach Gardens, FL, USA; <sup>d</sup>Center for Neurodegeneration and Experimental Therapeutics, University of Alabama at Birmingham, Birmingham, USA</footnote>

<footnote><sup>a</sup>MRC Prion Unit at UCL, UCL Institute of Prion Diseases, Courtauld Building, London, UK</footnote>
<figure><img src="image_72.png" /></figure>

variation regarding prion protein glycosylation. The analysis was performed on VRQ/VRQ sheep brain affected with classical scrapie. The ovine PrP<sup>Sc</sup> was isolated and purified by immunopurification and SDS-PAGE. The isolation of the scrapie prion protein was shown to be somewhat challenging, since glycoproteins are heterogeneously glycosylated, this leads to a decreased concentration of each analyte (each specific glycan). After obtaining a sufficient amount and purifying the protein, the di- and monoglycosylated forms of the prion protein were extracted from the gel and in-gel trypsin digestion was performed. The tryptic glycopeptides were then analysed using reverse phase liquid chromatography coupled with electrospray mass spectrometry (LC-ESI-MS). The glycan structures found on the ovine PrP<sup>Sc</sup> have been shown to contain 'brain-specific' N-glycans, which are high in fucose and sialic acids. Other features found, that are characteristic for brain N-glycans, include hybrid-type structures, bisecting N-Acetylglucosamine (GlcNAc) and core (α1-6)fucose, as well as outer arm (α1-3)fucose. We were able to develop a method for isolation of ovine PrP<sup>Sc</sup> from infected tissue and analysing the glycopeptides with LC-ESI-MS. With this approach we plan to compare glycan structures between different ovine prion strains in order to better understand whether or not N-glycans contribute to prion strain differences.

**KEYWORDS:** Prion; N-glycan; LC-ESI-MS

**110. Involvement of MAPK pathway and tau in pα-syn*-induced mitotoxicity**

Diego Grassi<sup>a,b</sup>, Natalia Diaz-Perez<sup>c</sup>, Laura A. Volpicelli-Daley<sup>d</sup> and Corinne Ida Lasmézas<sup>a,b</sup>

**CONTACT** Corinne Ida Lasmézas lasmezas@scripps.edu

**ABSTRACT**

We recently identified a phosphorylated form of α-synuclein, pα-syn*, as a key neurotoxic α-synuclein species found in cultured neurons, as well as in mouse and Parkinson's disease patients' brains. Small pα-syn* aggregates localize to mitochondria and induce mitochondrial damage and fragmentation. Herein, we investigated the molecular basis of pα-syn*-induced toxicity. By immunofluorescence, we found phosphorylated MKK4, JNK, ERK5 and p38 MAPKs in pα-syn* inclusions. pJNK colorized with pα-syn* at mitochondria and mitochondria-

associated ER membranes where it was associated with BiP and pACC1. We also found that pα-syn* aggregates are tightly associated with small ptau aggregates of similar size. Pα-syn*/ptau inclusions localized to areas of mitochondrial damage and to mitophagic vesicles, showing their role in mitochondrial toxicity, mitophagy induction, and their removal along with damaged mitochondrial fragments. Several MAPKs may act cooperatively to phosphorylate tau, notably JNK, p38, and GSK3β, a non-MAPK that was also found phosphorylated in the vicinity of pα-syn*/ptau aggregates. Thus, pα-syn* appears to be the trigger of a series of kinase mediated pathogenic events and a link between α-syn pathology and tau, another protein known to aggregate in Parkinson's disease and other synucleinopathies.

**KEYWORDS:** Alpha-synuclein; tau; Parkinson's disease; primary neurons; kinases; neurotoxicity; mitochondria; mitophagy

**111. Identification of rare genetic variants associated with sporadic CJD by exome sequencing**

Helen Speedy<sup>a</sup>, Penny Norsworthy<sup>a</sup>, Holger Hummerich<sup>a</sup>, Gary Adamson<sup>a</sup>, James Uphill<sup>a</sup>, John Collinge<sup>a</sup> and Simon Mead<sup>a</sup>

**CONTACT** Helen Speedy h.speedy@ucl.ac.uk

**ABSTRACT**

**Background:** Prion diseases are progressive neurodegenerative conditions characterized by the misfolding and aggregation of prion protein. Sporadic Creutzfeldt-Jakob Disease (sCJD) is the most common human prion disease. The common polymorphism at codon 129 of the *PRNP* gene influences sCJD risk; however additional genetic factors are thought to contribute to disease susceptibility. Ongoing genome-wide association studies have identified common genetic variants associated with sCJD risk. In this study we use exome sequencing to investigate the role of rare, disruptive variants as determinants of susceptibility to sCJD.

**Materials and Methods:** This study incorporates exome sequencing of an in-house set of 249 sCJD cases as well as data from 126 sCJD cases from the Medical Research Council (MRC) Brain Bank Network. Controls are derived from publicly available resources.

In-house, genomic DNA was extracted from peripheral blood. Exome capture using Agilent technology and paired-end sequencing on an Illumina HiSeq2000 sequencer was performed at Source Bioscience (Nottingham, UK).

<footnote><sup>a</sup>Department of Immunology and Microbiology, The Scripps Research Institute, Scripps Florida, Jupiter, FL, USA; <sup>b</sup>Department of Neuroscience, The Scripps Research Institute, Scripps Florida, Jupiter, FL, USA; <sup>c</sup>Private address, Palm Beach Gardens, FL, USA; <sup>d</sup>Center for Neurodegeneration and Experimental Therapeutics, University of Alabama at Birmingham, Birmingham, USA</footnote>

<footnote><sup>a</sup>MRC Prion Unit at UCL, UCL Institute of Prion Diseases, Courtauld Building, London, UK</footnote>
<figure><img src="image_73.png" /></figure>

Sequencing reads were aligned to the human reference genome (hg19) using Burrows-Wheeler Aligner software. Alignments were processed using the Genome Analysis Tool Kit pipeline, according to best practices. Variants were filtered according to quality and frequency metrics, as well as predicted functional consequences. Single variant and gene-based association testing was performed.

**Results:** Initial analyses of exome sequencing data failed to detect a single rare, disruptive genetic variant significantly associated with sCJD risk, whilst no individual gene in burden testing surpassed genome-wide significance. Future work will focus on exploring alternative rare variant analysis methods and the subsequent prioritization of variants for validation and follow-up in additional sCJD cases. In conjunction, we will examine the burden of clinically relevant (novel or known pathogenic) mutations in known dementia genes, deriving candidate genes from the MRC Dementia Gene Panel, UK Genetic Testing Network Clinical Panels and through manual literature curation.

**Conclusions:** We have conducted an exome sequencing study involving 375 sCJD cases to examine the role of rare, disruptive genetic variants in sCJD risk. Although no single variant or gene of significant impact was discovered in initial analyses, further exploration of this dataset is ongoing.

**KEYWORDS:** Genetics; exome; rare variant; sporadic Creutzfeldt-Jakob disease

## 112. The low levels of nerve growth factor and its upstream regulatory kinases in prion infection is reversed by resveratrol

Chao Hu<sup>a,b*</sup>, Cao Chen<sup>b,c*</sup>, Jia Chen<sup>a</sup>, Kang Xiao<sup>b,c</sup>, Jing Wang<sup>b,c</sup>, Qi Shi<sup>b,c</sup>, Yue Ma<sup>b</sup>, Li-Ping Gao<sup>b</sup>, Yue-Zhang Wu<sup>b</sup>, Lian Liu<sup>a</sup>, Ying Xia<sup>a</sup>, Pu Yan<sup>a</sup>, Adalaiti Maimaitiming<sup>b</sup>, Dong-Hua Zhou<sup>b</sup>, Li-Na Zhang<sup>b</sup>, Zhi-Bao Chen<sup>a</sup> and Xiao-Ping Dong<sup>b,c,d</sup>

CONTACT C. Chen chencao@ivdc.chinacdc.cn; Z. B. Chen chenzhibao_2000@sina.com; X. P. Dong dongxp238@sina.com

* These authors contributes equally to this work.

### ABSTRACT

Up to the present, none of the specific vaccines or therapeutic drugs has been documented to be

effective in human or animal prion diseases. Recently, resveratrol shows the ability to eliminate prion replication. To obtain insights into the possible changes of brain neurotrophic factor (NGF) and its upstream regulatory cascade during prion infection and after removal of prion propagation, the levels of NGF and its upstream regulatory factors in various SMB cell lines and in the mice brains inoculated with various SMB cellular lysates were assessed with various methodologies. The levels of NGF were significantly decreased *in vivo* and *in vitro*, while recovered after removal of PrP<sup>Sc</sup> by resveratrol. Morphological assays revealed that the NGF signals mainly colocalized within neurons, but not in the proliferative astrocytes and microglia. The upstream positive regulatory kinases, such as p-CREB, p-CaMKIV, CaMKK2 were also remarkably decreased in the prion infected cells and mice brains, whereas the negative regulatory one, p-CaMKK2, was increased. The aberrant situations of those kinases in prion infected cell lines or mice brains could be also partially reversed by removal of prion agent. Moreover, we demonstrated that the signals of CaMKK2 and p-CaMKK2 were also distributed predominately in neurons in the brain tissues. The data here illustrate a direct linkage of abnormally repressive NGF and its upstream regulatory kinases with prion infection. Resveratrol has not only the ability to inhibit prion replication, but also to improve the expression of NGF via CaMKK2/CaMKIV cascade, which might benefit the microenvironment in brains.

**KEYWORDS:** Prion; resveratrol; NGF; CaMKIV; CaMKK2

## 113. Diagnosis of CWD in a herd of farmed red deer

Ines Walther, Antanas Staskevicius, Andrei Soutyrine and Gordon Mitchell

National & OIE Reference Laboratory for Scrapie and CWD, Canadian Food Inspection Agency, Ottawa, ON, Canada

CONTACT Gordon Mitchell gordon.mitchell2@canada.ca

### ABSTRACT

Chronic wasting disease (CWD) continues to adversely impact wild and farmed cervids of North America, affecting primarily white-tailed deer (*Odocoileus virginianus*), mule deer (*Odocoileus hemionus*), and elk (*Cervus canadensis*). Red deer (*Cervus elaphus*) are closely related to elk, and occasional cases of CWD have been reported

<footnote><sup>a</sup>College of Life Science and Technology, Heilongjiang Bayi Agricultural University, Daqing, China; <sup>b</sup>State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China; <sup>c</sup>Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, China; <sup>d</sup>Center for Global Public Health, Chinese Center for Disease Control and Prevention, Beijing, China</footnote>
<figure><img src="image_73.png" /></figure>

Sequencing reads were aligned to the human reference genome (hg19) using Burrows-Wheeler Aligner software. Alignments were processed using the Genome Analysis Tool Kit pipeline, according to best practices. Variants were filtered according to quality and frequency metrics, as well as predicted functional consequences. Single variant and gene-based association testing was performed.

**Results:** Initial analyses of exome sequencing data failed to detect a single rare, disruptive genetic variant significantly associated with sCJD risk, whilst no individual gene in burden testing surpassed genome-wide significance. Future work will focus on exploring alternative rare variant analysis methods and the subsequent prioritization of variants for validation and follow-up in additional sCJD cases. In conjunction, we will examine the burden of clinically relevant (novel or known pathogenic) mutations in known dementia genes, deriving candidate genes from the MRC Dementia Gene Panel, UK Genetic Testing Network Clinical Panels and through manual literature curation.

**Conclusions:** We have conducted an exome sequencing study involving 375 sCJD cases to examine the role of rare, disruptive genetic variants in sCJD risk. Although no single variant or gene of significant impact was discovered in initial analyses, further exploration of this dataset is ongoing.

**KEYWORDS:** Genetics; exome; rare variant; sporadic Creutzfeldt-Jakob disease

## 112. The low levels of nerve growth factor and its upstream regulatory kinases in prion infection is reversed by resveratrol

Chao Hu<sup>a,b*</sup>, Cao Chen<sup>b,c*</sup>, Jia Chen<sup>a</sup>, Kang Xiao<sup>b,c</sup>, Jing Wang<sup>b,c</sup>, Qi Shi<sup>b,c</sup>, Yue Ma<sup>b</sup>, Li-Ping Gao<sup>b</sup>, Yue-Zhang Wu<sup>b</sup>, Lian Liu<sup>a</sup>, Ying Xia<sup>a</sup>, Pu Yan<sup>a</sup>, Adalaiti Maimaitiming<sup>b</sup>, Dong-Hua Zhou<sup>b</sup>, Li-Na Zhang<sup>b</sup>, Zhi-Bao Chen<sup>a</sup> and Xiao-Ping Dong<sup>b,c,d</sup>

CONTACT C. Chen chencao@ivdc.chinacdc.cn; Z. B. Chen chenzhibao_2000@sina.com; X. P. Dong dongxp238@sina.com

* These authors contributes equally to this work.

### ABSTRACT

Up to the present, none of the specific vaccines or therapeutic drugs has been documented to be

effective in human or animal prion diseases. Recently, resveratrol shows the ability to eliminate prion replication. To obtain insights into the possible changes of brain neurotrophic factor (NGF) and its upstream regulatory cascade during prion infection and after removal of prion propagation, the levels of NGF and its upstream regulatory factors in various SMB cell lines and in the mice brains inoculated with various SMB cellular lysates were assessed with various methodologies. The levels of NGF were significantly decreased *in vivo* and *in vitro*, while recovered after removal of PrP<sup>Sc</sup> by resveratrol. Morphological assays revealed that the NGF signals mainly colocalized within neurons, but not in the proliferative astrocytes and microglia. The upstream positive regulatory kinases, such as p-CREB, p-CaMKIV, CaMKK2 were also remarkably decreased in the prion infected cells and mice brains, whereas the negative regulatory one, p-CaMKK2, was increased. The aberrant situations of those kinases in prion infected cell lines or mice brains could be also partially reversed by removal of prion agent. Moreover, we demonstrated that the signals of CaMKK2 and p-CaMKK2 were also distributed predominately in neurons in the brain tissues. The data here illustrate a direct linkage of abnormally repressive NGF and its upstream regulatory kinases with prion infection. Resveratrol has not only the ability to inhibit prion replication, but also to improve the expression of NGF via CaMKK2/CaMKIV cascade, which might benefit the microenvironment in brains.

**KEYWORDS:** Prion; resveratrol; NGF; CaMKIV; CaMKK2

## 113. Diagnosis of CWD in a herd of farmed red deer

Ines Walther, Antanas Staskevicius, Andrei Soutyrine and Gordon Mitchell

National & OIE Reference Laboratory for Scrapie and CWD, Canadian Food Inspection Agency, Ottawa, ON, Canada

CONTACT Gordon Mitchell gordon.mitchell2@canada.ca

### ABSTRACT

Chronic wasting disease (CWD) continues to adversely impact wild and farmed cervids of North America, affecting primarily white-tailed deer (*Odocoileus virginianus*), mule deer (*Odocoileus hemionus*), and elk (*Cervus canadensis*). Red deer (*Cervus elaphus*) are closely related to elk, and occasional cases of CWD have been reported

<footnote><sup>a</sup>College of Life Science and Technology, Heilongjiang Bayi Agricultural University, Daqing, China; <sup>b</sup>State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China; <sup>c</sup>Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, China; <sup>d</sup>Center for Global Public Health, Chinese Center for Disease Control and Prevention, Beijing, China</footnote>
<figure><img src="image_74.png" /></figure>

in farmed red deer in the United States and the Republic of Korea, and in a wild red deer in Norway. CWD was recently detected in farmed red deer for the first time in Canada, in a region geographically distinct from all previous cases of CWD. Understanding the diagnostic features and pathogenesis of CWD in red deer is essential to developing effective disease detection and control strategies in this species. The index case of this herd, a clinically healthy 15-month old male red deer, was initially detected during routine slaughter surveillance testing by ELISA. Confirmatory testing by immunohistochemistry (IHC) demonstrated modest aggregates of pathological prion protein (PrPCWD) in restricted regions of the obex of the medulla and retropharyngeal lymph nodes. Western blot of the obex homogenate revealed a banding pattern consistent with that of other Canadian CWD cases, and distinct from what has been observed in red deer experimentally infected with bovine spongiform encephalopathy. Subsequent disease control measures in this herd resulted in the testing of over 1700 red deer and an additional 10 cases of CWD were identified in females ranging in age from 18 to 28 months. All cases were positive in the obex by ELISA, IHC, and Western blot, although the extent of PrPCWD deposition detected by IHC was somewhat variable. A higher degree of variability in PrPCWD accumulation was found in lymphoid tissues, with PrPCWD not detectable by IHC in the tonsils of several cases. Sequencing of the prion protein gene from all positive cases and a subset of negative cases identified variability at several codons (98, 168, and 226) although the extent to which these polymorphisms may influence susceptibility to infection is not yet evident. Natural transmission of CWD occurs relatively efficiently amongst cervids, facilitating the geographical dissemination of disease to previously naive populations. Determining the unique characteristics of CWD in different cervid species is needed to inform the refinement of disease management approaches.

## 114. Structural analysis of human and transgenic mouse-derived tau protein aggregates

Shelaine C. Fleck<sup>a,b</sup>, Ghazaleh Eskandari-Sedighi<sup>a,b</sup>, Razieh Kamali-Jamil<sup>a,b</sup>, Mirka Kasirova<sup>c</sup>, Chae Kim<sup>c</sup>, Howard Young<sup>b</sup>, David Westaway<sup>a,b,d</sup>, Jiri Safar<sup>c</sup> and Holger Wille<sup>a,b</sup>

CONTACT Shelaine C. Fleck [shelaine@ualberta.ca](mailto:shelaine@ualberta.ca)

## ABSTRACT

Tauopathies are a class of neurological disorders associated with the aggregation of the tau protein into neurofibrillary tangles. The most prominent tauopathy is Alzheimer's disease (AD), which presents as two forms: early onset (genetic, gAD) and late onset (sporadic, sAD). sAD does not have a known cause; although environmental, lifestyle, and genetic factors are thought to contribute to the disease manifestation. gAD can be caused by one of three different single gene mutations: in the amyloid precursor protein (APP), presenilin 1, or presenilin 2. In both forms of AD, the tau protein aggregates to form intracellular fibrillary tangles, which spread along neuronal networks. Amyloid-β, the product of APP proteolysis, also contributes to AD through the formation of extracellular amyloid plaques. Together, these proteins lead to the extensive neuronal death seen in AD. The structure of tau fibrils in AD, either as Paired Helical Filaments (PHFs) or Straight Filaments (SFs), was solved using cryo-electron microscopy and 3D-reconstructions.¹ By analysing PHFs and SFs from sAD, researchers found that filament cores consist of two identical protofilaments which adopt a cross-β structure [1]. Later, researchers analysed additional samples from sAD and gAD cases, which confirmed the initial findings [2]. Other tauopathies, including Pick's disease and frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), are caused solely by the misfolding of the tau protein. Each of these diseases is characterized by the dementia they cause and notable brain atrophy. The structure of tau fibrils in Pick's disease accumulating predominantly three-repeat tau differs from AD, it forms a single protofilament with nine beta-strands [3]. The structure of tau fibrils in FTDP-17 is currently unknown. The focus of our research is to further investigate the different conformations of the tau protein in these diseases. Using EM and 3D-reconstructions we plan to examine samples from gAD and sAD cases. Currently, we have examined samples from rapidly progressive AD and a transgenic mouse line expressing four-repeat human tau protein with a P301L mutation, which is found in some FTDP-17 patients [4]. Previously, three tau fibril morphologies: straight, coiled, and twisted-ribbon, were identified [4]. Through continued examination of these fibrils using 3D-reconstructions and volumetric visualization, we hope to see more detailed differences in the structure adopted by the tau protein. This will provide insights into the role the tau protein plays in these tauopathies and how the structural differences may contribute to the distinct disease manifestations.

## References

[1] Fitzpatrick, et al. Nature. 2017;547:185-190.

<footnote><sup>a</sup>Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, AB, Canada; <sup>b</sup>Department of Biochemistry, University of Alberta, Edmonton, AB, Canada; <sup>c</sup>Departments of Pathology and Neurology, Case Western Reserve University, Cleveland, OH, USA; <sup>d</sup>Department of Medicine, University of Alberta, Edmonton, AB, Canada</footnote>
<figure><img src="image_74.png" /></figure>

in farmed red deer in the United States and the Republic of Korea, and in a wild red deer in Norway. CWD was recently detected in farmed red deer for the first time in Canada, in a region geographically distinct from all previous cases of CWD. Understanding the diagnostic features and pathogenesis of CWD in red deer is essential to developing effective disease detection and control strategies in this species. The index case of this herd, a clinically healthy 15-month old male red deer, was initially detected during routine slaughter surveillance testing by ELISA. Confirmatory testing by immunohistochemistry (IHC) demonstrated modest aggregates of pathological prion protein (PrPCWD) in restricted regions of the obex of the medulla and retropharyngeal lymph nodes. Western blot of the obex homogenate revealed a banding pattern consistent with that of other Canadian CWD cases, and distinct from what has been observed in red deer experimentally infected with bovine spongiform encephalopathy. Subsequent disease control measures in this herd resulted in the testing of over 1700 red deer and an additional 10 cases of CWD were identified in females ranging in age from 18 to 28 months. All cases were positive in the obex by ELISA, IHC, and Western blot, although the extent of PrPCWD deposition detected by IHC was somewhat variable. A higher degree of variability in PrPCWD accumulation was found in lymphoid tissues, with PrPCWD not detectable by IHC in the tonsils of several cases. Sequencing of the prion protein gene from all positive cases and a subset of negative cases identified variability at several codons (98, 168, and 226) although the extent to which these polymorphisms may influence susceptibility to infection is not yet evident. Natural transmission of CWD occurs relatively efficiently amongst cervids, facilitating the geographical dissemination of disease to previously naive populations. Determining the unique characteristics of CWD in different cervid species is needed to inform the refinement of disease management approaches.

## 114. Structural analysis of human and transgenic mouse-derived tau protein aggregates

Shelaine C. Fleck<sup>a,b</sup>, Ghazaleh Eskandari-Sedighi<sup>a,b</sup>, Razieh Kamali-Jamil<sup>a,b</sup>, Mirka Kasirova<sup>c</sup>, Chae Kim<sup>c</sup>, Howard Young<sup>b</sup>, David Westaway<sup>a,b,d</sup>, Jiri Safar<sup>c</sup> and Holger Wille<sup>a,b</sup>

CONTACT Shelaine C. Fleck [shelaine@ualberta.ca](mailto:shelaine@ualberta.ca)

## ABSTRACT

Tauopathies are a class of neurological disorders associated with the aggregation of the tau protein into neurofibrillary tangles. The most prominent tauopathy is Alzheimer's disease (AD), which presents as two forms: early onset (genetic, gAD) and late onset (sporadic, sAD). sAD does not have a known cause; although environmental, lifestyle, and genetic factors are thought to contribute to the disease manifestation. gAD can be caused by one of three different single gene mutations: in the amyloid precursor protein (APP), presenilin 1, or presenilin 2. In both forms of AD, the tau protein aggregates to form intracellular fibrillary tangles, which spread along neuronal networks. Amyloid-β, the product of APP proteolysis, also contributes to AD through the formation of extracellular amyloid plaques. Together, these proteins lead to the extensive neuronal death seen in AD. The structure of tau fibrils in AD, either as Paired Helical Filaments (PHFs) or Straight Filaments (SFs), was solved using cryo-electron microscopy and 3D-reconstructions.¹ By analysing PHFs and SFs from sAD, researchers found that filament cores consist of two identical protofilaments which adopt a cross-β structure [1]. Later, researchers analysed additional samples from sAD and gAD cases, which confirmed the initial findings [2]. Other tauopathies, including Pick's disease and frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), are caused solely by the misfolding of the tau protein. Each of these diseases is characterized by the dementia they cause and notable brain atrophy. The structure of tau fibrils in Pick's disease accumulating predominantly three-repeat tau differs from AD, it forms a single protofilament with nine beta-strands [3]. The structure of tau fibrils in FTDP-17 is currently unknown. The focus of our research is to further investigate the different conformations of the tau protein in these diseases. Using EM and 3D-reconstructions we plan to examine samples from gAD and sAD cases. Currently, we have examined samples from rapidly progressive AD and a transgenic mouse line expressing four-repeat human tau protein with a P301L mutation, which is found in some FTDP-17 patients [4]. Previously, three tau fibril morphologies: straight, coiled, and twisted-ribbon, were identified [4]. Through continued examination of these fibrils using 3D-reconstructions and volumetric visualization, we hope to see more detailed differences in the structure adopted by the tau protein. This will provide insights into the role the tau protein plays in these tauopathies and how the structural differences may contribute to the distinct disease manifestations.

## References

[1] Fitzpatrick, et al. Nature. 2017;547:185-190.

<footnote><sup>a</sup>Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, AB, Canada; <sup>b</sup>Department of Biochemistry, University of Alberta, Edmonton, AB, Canada; <sup>c</sup>Departments of Pathology and Neurology, Case Western Reserve University, Cleveland, OH, USA; <sup>d</sup>Department of Medicine, University of Alberta, Edmonton, AB, Canada</footnote>
<figure><img src="image_75.png" /></figure>

[2] Falcon, et al. Acta Neuropathol. 2018;136:699-708.

[3] Falcon, et al. Nature. 2018;561:137-140.

[4] Eskandari-Sedighi, et al. Mol Neurodegener. 2017;12:72.

## 115. Diversity of chronic wasting disease prion strains

Camilo Duque Velásquezª, Elizabeth Triscottª, Chiye Kimª, Jacques Van der Merweª, Samia Hannaouiᵇ, Trent Bollingerᶜ, Christina Carlsonᵈ, Sylvie Benestadᵉ, Sabine Gilchᵇ, Judd Aikenf and Debbie McKenzieª

ªDepartment of Biological Sciences, Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Canada;
ᵇDepartment of Ecosystem and Public Health, Calgary Prion Research Unit, University of Calgary, Calgary, Canada;
ᶜDepartment of Veterinary Pathology, Canadian Wildlife Health Cooperative, University of Saskatchewan, Saskatoon, Canada; ᵈU.S. Geological Survey-National Wildlife Health Center, Madison, WI, USA;
ᵉNorwegian Veterinary Institute, Oslo and Trondheim, Norway;
fDepartment of Agricultural, Food and Nutritional Sciences, Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Canada

### ABSTRACT

Chronic Wasting Disease (CWD) prions affect a variety of cervid species. The expansion of the CWD geographic range and the increasing prevalence makes CWD a concern for wildlife, livestock, and human health. Prion pathogenesis results from the template-directed misfolding of cellular prion proteins (PrP<sup>C</sup>) into conformational species (e.g. PrP<sup>CWD</sup>) associated with distinct strains. We propose that cervid cellular prion protein (PrP<sup>C</sup>) polymorphisms have resulted in prion conformational diversification and speciation (i.e. by adaptive radiation) resulting in emergence of novel CWD strains. New prion strain conformers emerge following transmission between cervids expressing different PrP<sup>C</sup> amino acid polymorphisms [1-4]. Emergent CWD strains can have novel transmission properties that enable them to infect host species previously considered resistant, suggesting an increase in zoonotic risk as strain-conformers diversify and evolve [2, 3]. We are comparing field CWD isolates of different cervid species from various regions of North America and Norway and have identified differences in biochemical properties of PrP<sup>CWD</sup>, in vitro and ex vivo propagation and transmission into transgenic mice expressing deer and elk PrP (tgDeer - 96G, tgDeer - 96S, and tgElk-E226) as well as C57Bl6 mice and hamsters. Variations in PrP-res type and protease sensitivity were observed following treatment with proteinase K. Comparison of the PMCA seeding activity using deer and elk PrP<sup>C</sup> as substrates revealed differences between elk CWD isolates. ELK21 cells also responded

differently to various cervid prions. Transmission was efficient for all isolates in tg33 and tgElk mice; however, only a few isolates were able to propagate in host expressing S96 deer PrP<sup>C</sup>, which has been shown to impose a strong transmission barrier, providing a means of differential selection of CWD strains [2]. Transmission differences were observed following interspecies transmission. While some white-tailed deer and mule deer isolates failed to transmit into hamsters, other isolates transmitted with a low attack rate but considerable sub-clinical infection. These isolates had a migration pattern similar to the hamster-passaged Wisc-1 strain. Two other isolates, transmitted more efficiently into hamsters and produced two different PrP-res migration profiles compared to Wisc-1-like PrP-res. Transmission of Norwegian moose and reindeer CWD isolates is ongoing; preliminary results will be presented. Our data indicate the existence of at least five different CWD strains based on transmission properties.

### References

[1] Johnson CJ, et al. Prion protein polymorphisms affect chronic wasting disease progression. PloS ONE. 2011;6 (3):e17450.

[2] Duque Velasquez C, 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. Emerging Infect Dis. 2017;23(9):1598-1600.

[4] Hannaoui S, et al. Destabilizing polymorphism in cervid prion protein hydrophobic core determines prion conformation and conversion efficiency. PloS Pathog. 2018;13(8):e1006553.

## 116. Characterization of distinct SOD1 prion strains

Jacob I. Ayers<sup>a,b</sup>, Kristy Dillon<sup>a</sup>, Jeffrey Diamond<sup>a</sup>, Eric Fagerli<sup>a</sup>, Susan Fromholt<sup>a</sup> and David R. Borchelt<sup>a</sup>

CONTACT Jacob I. Ayers jacob.ayers@ucsf.edu

### ABSTRACT

Mutations in the Cu-Zn superoxide dismutase 1 (SOD1) gene are known to be causative in 10-20% of familial amyotrophic lateral sclerosis (fALS) cases. The duration of disease in these patients can vary, such that some mutations are associated with disease of less than

<footnote>ªDepartment of Neuroscience, Center for Translational Research in Neurodegenerative Disease, University of Florida, Gainesville, FL, USA;
ᵇDepartment of Neurology, Institute for Neurodegenerative Diseases, University of California San Francisco, San Francisco, CA, USA</footnote>
<figure><img src="image_75.png" /></figure>

[2] Falcon, et al. Acta Neuropathol. 2018;136:699-708.

[3] Falcon, et al. Nature. 2018;561:137-140.

[4] Eskandari-Sedighi, et al. Mol Neurodegener. 2017;12:72.

## 115. Diversity of chronic wasting disease prion strains

Camilo Duque Velásquezª, Elizabeth Triscottª, Chiye Kimª, Jacques Van der Merweª, Samia Hannaouiᵇ, Trent Bollingerᶜ, Christina Carlsonᵈ, Sylvie Benestadᵉ, Sabine Gilchᵇ, Judd Aikenf and Debbie McKenzieª

ªDepartment of Biological Sciences, Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Canada;
ᵇDepartment of Ecosystem and Public Health, Calgary Prion Research Unit, University of Calgary, Calgary, Canada;
ᶜDepartment of Veterinary Pathology, Canadian Wildlife Health Cooperative, University of Saskatchewan, Saskatoon, Canada; ᵈU.S. Geological Survey-National Wildlife Health Center, Madison, WI, USA;
ᵉNorwegian Veterinary Institute, Oslo and Trondheim, Norway;
fDepartment of Agricultural, Food and Nutritional Sciences, Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Canada

### ABSTRACT

Chronic Wasting Disease (CWD) prions affect a variety of cervid species. The expansion of the CWD geographic range and the increasing prevalence makes CWD a concern for wildlife, livestock, and human health. Prion pathogenesis results from the template-directed misfolding of cellular prion proteins (PrPC) into conformational species (e.g. PrPCWD) associated with distinct strains. We propose that cervid cellular prion protein (PrPC) polymorphisms have resulted in prion conformational diversification and speciation (i.e. by adaptive radiation) resulting in emergence of novel CWD strains. New prion strain conformers emerge following transmission between cervids expressing different PrPC amino acid polymorphisms [1-4]. Emergent CWD strains can have novel transmission properties that enable them to infect host species previously considered resistant, suggesting an increase in zoonotic risk as strain-conformers diversify and evolve [2, 3]. We are comparing field CWD isolates of different cervid species from various regions of North America and Norway and have identified differences in biochemical properties of PrPCWD, in vitro and ex vivo propagation and transmission into transgenic mice expressing deer and elk PrP (tgDeer - 96G, tgDeer - 96S, and tgElk-E226) as well as C57Bl6 mice and hamsters. Variations in PrP-res type and protease sensitivity were observed following treatment with proteinase K. Comparison of the PMCA seeding activity using deer and elk PrPC as substrates revealed differences between elk CWD isolates. ELK21 cells also responded

differently to various cervid prions. Transmission was efficient for all isolates in tg33 and tgElk mice; however, only a few isolates were able to propagate in host expressing S96 deer PrPC, which has been shown to impose a strong transmission barrier, providing a means of differential selection of CWD strains [2]. Transmission differences were observed following interspecies transmission. While some white-tailed deer and mule deer isolates failed to transmit into hamsters, other isolates transmitted with a low attack rate but considerable sub-clinical infection. These isolates had a migration pattern similar to the hamster-passaged Wisc-1 strain. Two other isolates, transmitted more efficiently into hamsters and produced two different PrP-res migration profiles compared to Wisc-1-like PrP-res. Transmission of Norwegian moose and reindeer CWD isolates is ongoing; preliminary results will be presented. Our data indicate the existence of at least five different CWD strains based on transmission properties.

### References

[1] Johnson CJ, et al. Prion protein polymorphisms affect chronic wasting disease progression. PloS ONE. 2011;6 (3):e17450.

[2] Duque Velasquez C, 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. Emerging Infect Dis. 2017;23(9):1598-1600.

[4] Hannaoui S, et al. Destabilizing polymorphism in cervid prion protein hydrophobic core determines prion conformation and conversion efficiency. PloS Pathog. 2018;13(8):e1006553.

## 116. Characterization of distinct SOD1 prion strains

Jacob I. Ayersª, Kristy Dillonª, Jeffrey Diamondª, Eric Fagerliª, Susan Fromholtª and David R. Borcheltª

ªDepartment of Neuroscience, Center for Translational Research in Neurodegenerative Disease, University of Florida, Gainesville, FL, USA;
ᵇDepartment of Neurology, Institute for Neurodegenerative Diseases, University of California San Francisco, San Francisco, CA, USA

CONTACT Jacob I. Ayers jacob.ayers@ucsf.edu

### ABSTRACT

Mutations in the Cu-Zn superoxide dismutase 1 (SOD1) gene are known to be causative in 10-20% of familial amyotrophic lateral sclerosis (fALS) cases. The duration of disease in these patients can vary, such that some mutations are associated with disease of less than
<figure><img src="image_76.png" /></figure>

3 years, whereas others are associated with disease of
more than 20 years. One potential explanation for this
variability in disease progression is that distinct SOD1
mutations exhibit conformational strain properties that
may affect their ability to propagate within the CNS. To
address this, we tested various preparations containing
different SOD1 mutations in *in vivo* and *ex vivo* seeding
assays to investigate several SOD1 properties. We
injected multiple misfolded SOD1-containing prepara-
tions within the spinal cords of mice at postnatal day
P0. These recipient mice express low levels of the G85R
variant of human SOD1 fused to YFP (G85R-SOD1:
YFP) and are permissive for motor neuron disease
induction. Once MND was induced, the spinal cords
were analysed for the presence and morphology of the
induced G85R-SOD1:YFP aggregates by direct fluores-
cence. Additionally, as an *ex vivo* assay, these SOD1-
containing preparations were added to organotypic
spinal cord slice cultures prepared from G85R-SOD1:
YFP mice to directly visualize the induced aggregates
over time. We observed both distinct SOD1:YFP aggre-
gate pathologies and incubation periods that bred true
upon repeated passages. Additionally, we observed the
same SOD1:YFP aggregate structures in spinal cord
slice cultures as was seen in mice, when treated with a
given SOD1 containing preparation. The distinct
SOD1:YFP pathologies observed suggest prion strain-
like properties for SOD1. Additionally, the fact that the
morphology of these structures bred true upon second
passage in mice with the same genotype suggests that
the formation of these aggregates are produced by
conformational templated misfolding, which is respon-
sible for the propagation of prions. These results
strongly suggest SOD1 prions are capable of exhibiting
conformational strain-like variability in the misfolding
and propagation of SOD1-linked ALS.

117. Bank vole bioassay for detection of prion infection in CWD-challenged cynomolgus macaques

Karla A. Schwenkeª, Joo-Hee Waelzleinª, Hermann M. Schatzlᵇ, c, Walter Schulz-Schaeffere, Christiane Stahl-Hennigf, Stefanie Czubc, d and Michael Beekesa

ªRobert Koch Institute, Prion and Prionoid Research Unit, ZBS 6, Berlin, Germany; ᵇUniversity of Calgary, Calgary Prion Research Unit, Calgary, Canada; ᶜUniversity of Calgary, Faculty of Veterinary Medicine, Calgary, Canada; ᵈCanadian Food Inspection Agency (CFIA), Lethbridge, Canada; ᵉUniversity of Homburg/Saar, Homburg, Germany; ṿGerman Primate Center, Goettingen, Germany

CONTACT Karla A. Schwenke SchwenkeK@rki.de WaelzleinJ@rki.de

ABSTRACT

Although Chronic Wasting Disease (CWD) was
detected in cervids in the late 1960s, the risk of CWD
transmission from cervids to humans is still puzzling.
The zoonotic potential which might emanate from
CWD was studied by several research groups using a
multitude of *in vitro* and *in vivo* approaches. These
studies provided evidence for a significant transmission
barrier for CWD between cervids and humans, but do
not allow the definite exclusion of such inter-species
transmission. Old- world monkeys such as macaques
are currently considered the most reliable animal model
for testing the zoonotic potential of CWD prions. Using
oral and parenteral transmission routes, our research
consortium challenged cynomolgus monkeys with
characterized CWD material starting 10 years ago. To
test the presence or absence of prion infection in these
macaques, we injected bank voles intracerebrally with
homogenized brain, spinal cord and spleen tissue from
monkeys sacrificed 5-7years after CWD inoculation.
Bank voles have been shown to be a universal acceptor
for prions derived from various species and were found
to be particularly susceptible to infection with CWD
prions. Therefore, in our study bank vole bioassays
provide a method of choice for the detection of prion
infection in the CWD-challenged cynomolgus mon-
keys. Potential prion disease of macaques will be
detected by assessment of prion-specific clinical signs
in bank voles. The clinical assessment includes weight
monitoring and routine observation of the animals
according to a clinical score sheet. Further, CNS and
spleen tissue will be examined *post mortem* for PrP<sup>res</sup>
by Western blot analysis. In case of ambiguous results,
we plan to perform second passage inoculations in
bank voles. Taken together, these studies are expected
to contribute to a definite evaluation of the zoonotic
risk of CWD.

118. Effect of route of infection and PRNP genotype on detection of preclinical infection using blood samples from BSE-infected sheep

M. K. F. Salamatª, O. Andreolettiᵇ, S. McCutcheonª,
A. R. A. Blancoᶜ, J. C. Mansonª, E. F. Houstonª

ªThe Roslin Institute, R(D)SVS, University of Edinburgh, Easter Bush, Midlothian, EH25 9RG, UK; ᵇUMR INRA ENVT 1225, Ecole Nationale Vétérinaire de Toulouse, Toulouse, France; ᶜComponents Development Laboratory, NHS Blood and Transplant, Cambridge, UK

CONTACT E. F. Houston Fiona.Houston@roslin.ed.ac.uk
<figure><img src="image_76.png" /></figure>

3 years, whereas others are associated with disease of
more than 20 years. One potential explanation for this
variability in disease progression is that distinct SOD1
mutations exhibit conformational strain properties that
may affect their ability to propagate within the CNS. To
address this, we tested various preparations containing
different SOD1 mutations in *in vivo* and *ex vivo* seeding
assays to investigate several SOD1 properties. We
injected multiple misfolded SOD1-containing prepara-
tions within the spinal cords of mice at postnatal day
P0. These recipient mice express low levels of the G85R
variant of human SOD1 fused to YFP (G85R-SOD1:
YFP) and are permissive for motor neuron disease
induction. Once MND was induced, the spinal cords
were analysed for the presence and morphology of the
induced G85R-SOD1:YFP aggregates by direct fluores-
cence. Additionally, as an *ex vivo* assay, these SOD1-
containing preparations were added to organotypic
spinal cord slice cultures prepared from G85R-SOD1:
YFP mice to directly visualize the induced aggregates
over time. We observed both distinct SOD1:YFP aggre-
gate pathologies and incubation periods that bred true
upon repeated passages. Additionally, we observed the
same SOD1:YFP aggregate structures in spinal cord
slice cultures as was seen in mice, when treated with a
given SOD1 containing preparation. The distinct
SOD1:YFP pathologies observed suggest prion strain-
like properties for SOD1. Additionally, the fact that the
morphology of these structures bred true upon second
passage in mice with the same genotype suggests that
the formation of these aggregates are produced by
conformational templated misfolding, which is respon-
sible for the propagation of prions. These results
strongly suggest SOD1 prions are capable of exhibiting
conformational strain-like variability in the misfolding
and propagation of SOD1-linked ALS.

117. Bank vole bioassay for detection of prion infection in CWD-challenged cynomolgus macaques

Karla A. Schwenkeª, Joo-Hee Waelzleinª, Hermann M. Schatzlᵇ, c, Walter Schulz-Schaeffere, Christiane Stahl-Hennigf, Stefanie Czubc, d and Michael Beekesa

ªRobert Koch Institute, Prion and Prionoid Research Unit, ZBS 6, Berlin, Germany; ᵇUniversity of Calgary, Calgary Prion Research Unit, Calgary, Canada; ᶜUniversity of Calgary, Faculty of Veterinary Medicine, Calgary, Canada; ᵈCanadian Food Inspection Agency (CFIA), Lethbridge, Canada; ᵉUniversity of Homburg/Saar, Homburg, Germany; ṿGerman Primate Center, Goettingen, Germany

CONTACT Karla A. Schwenke SchwenkeK@rki.de WaelzleinJ@rki.de

ABSTRACT

Although Chronic Wasting Disease (CWD) was
detected in cervids in the late 1960s, the risk of CWD
transmission from cervids to humans is still puzzling.
The zoonotic potential which might emanate from
CWD was studied by several research groups using a
multitude of *in vitro* and *in vivo* approaches. These
studies provided evidence for a significant transmission
barrier for CWD between cervids and humans, but do
not allow the definite exclusion of such inter-species
transmission. Old- world monkeys such as macaques
are currently considered the most reliable animal model
for testing the zoonotic potential of CWD prions. Using
oral and parenteral transmission routes, our research
consortium challenged cynomolgus monkeys with
characterized CWD material starting 10 years ago. To
test the presence or absence of prion infection in these
macaques, we injected bank voles intracerebrally with
homogenized brain, spinal cord and spleen tissue from
monkeys sacrificed 5-7years after CWD inoculation.
Bank voles have been shown to be a universal acceptor
for prions derived from various species and were found
to be particularly susceptible to infection with CWD
prions. Therefore, in our study bank vole bioassays
provide a method of choice for the detection of prion
infection in the CWD-challenged cynomolgus mon-
keys. Potential prion disease of macaques will be
detected by assessment of prion-specific clinical signs
in bank voles. The clinical assessment includes weight
monitoring and routine observation of the animals
according to a clinical score sheet. Further, CNS and
spleen tissue will be examined *post mortem* for PrP<sup>res</sup>
by Western blot analysis. In case of ambiguous results,
we plan to perform second passage inoculations in
bank voles. Taken together, these studies are expected
to contribute to a definite evaluation of the zoonotic
risk of CWD.

118. Effect of route of infection and PRNP genotype on detection of preclinical infection using blood samples from BSE-infected sheep

M. K. F. Salamatª, O. Andreolettiᵇ, S. McCutcheonª,
A. R. A. Blancoᶜ, J. C. Mansonª, E. F. Houstonª

ªThe Roslin Institute, R(D)SVS, University of Edinburgh, Easter Bush, Midlothian, EH25 9RG, UK; ᵇUMR INRA ENVT 1225, Ecole Nationale Vétérinaire de Toulouse, Toulouse, France; ᶜComponents Development Laboratory, NHS Blood and Transplant, Cambridge, UK

CONTACT E. F. Houston Fiona.Houston@roslin.ed.ac.uk
<figure><img src="image_77.png" /></figure>

## ABSTRACT

The possibility of on-going blood-borne transmission of variant Creutzfeldt-Jakob disease (vCJD) is a major concern for public health authorities, particularly in light of uncertainties surrounding the prevalence of subclinical vCJD infection in the human population. A robust and highly sensitive assay that can be applied to readily accessible samples (e.g. blood, urine), for detection of infected individuals before the onset of disease, would be of enormous benefit in preventing further transmission of vCJD. Using sheep experimentally infected with BSE as a model, we have previously demonstrated that the protein misfolding amplification assay (PMCA) can detect seeding activity in blood samples as early as 6 months post-infection. However, we also observed significant differences in incubation period, and transmission of infection by transfusion, associated with genetic variation at *PRNP* codon 141¹. The aim of this study was to determine the relationship between codon 141 PRNP genotype and the onset of detection of seeding activity in preclinical blood samples from orally-infected (donor) and intravenously-infected (recipient) sheep.

Samples were analysed using a microplate-based, miniaturized bead serial PMCA protocol²⁻³, with brain homogenate from tgShpXI transgenic mice expressing sheep ARQ PrP (with added dextran sulphate) as substrate, and undiluted buffy coat samples from BSE-infected and uninfect ed sheep as seed. In orally challenged sheep, blood samples were collected at intervals from 2 months post-infection until the onset of clinical signs. The first time point at which blood samples tested positive was very variable, ranging from 4 months to 12 months post-infection and there was no obvious correlation between codon 141 genotype and the onset of detection. In sheep infected by blood transfusion, the first preclinical blood samples were collected at 6 months post-infection, and majority of animals tested gave positive PMCA results at this time point, regardless of codon 141 *PRNP* genotype. For both orally and intravenously-infected sheep, all samples collected at time points following the onset of detection tested positive in the PMCA. The results demonstrate that the route of infection appears to have a much more significant influence than *PRNP* genotype on the onset of detection of seeding activity in preclinical blood samples. They also demonstrate that once seeding activity is detected in blood samples from infected individuals, it is consistently detected in samples from subsequent time points throughout the incubation period. This data provides valuable insights into the factors that may influence the sensitivity of detection of preclinical prion infection using blood-based assays.

## References

[1] Tan, et al. J Gen Virol. 2012;93(Pt 12): 2749-2756

[2] Moudjou, et al. MBio. 2013 31;5(1):e00829-13

[3] Lacroux, et al. PLoS Pathog. 2014;10:e1004202.

## 119. Host genetic factors impacting prion diseases: genomic analysis of phenotypically distinct bovine spongiform encephalopathy cattle

Sandor Dudas<sup>a,b</sup>, Graham Plastow<sup>c</sup>, James Cross<sup>b</sup> and Stefanie Czub<sup>a,b</sup>

<sup>a</sup>Canadian Food Inspection Agency, National Center for Animal Disease Lethbridge; <sup>b</sup>University of Calgary, Faculty of Veterinary Medicine; <sup>c</sup>University of Alberta, Agriculture, Food and Nutritional Science

CONTACT Sandor Dudas sandor.dudas@canada.ca

Populations and development of diagnostics and effective therapeutics for AD.

## ABSTRACT

**Background/Introduction:** Bovine spongiform encephalopathy (BSE)-challenged cattle have variable susceptibility, incubation times and in rare cases, even different disease phenotypes; our initial results suggest this may be related to host genetic factors. Attempts have been made to link bovine spongiform encephalopathy susceptibility with genotype using BSE field cases. These sample populations have a number of uncontrolled variables making analysis difficult and likely contribute to a lack of agreement between studies.

**Materials and Methods:** To identify genetic factors impacting BSE in a more controlled population, tissue samples from BSE-challenged cattle have been collected from the APHA (UK), the FLI (Germany), the USDA (USA), and the CFIA (Canada). DNA from these tissues are currently being genotyped to identify regions of the bovine genome which are associated with different BSE phenotypes. Once identified, targeted next generation sequencing will be used to find functionally relevant genetic changes. If important polymorphisms are found, gene-edited cell culture will be used to demonstrate the impacts these genetic variants have on prion expression and disease progression.

**Results:** Genotyping of short incubation BSE challenge cattle shows a significant link with the deletion genotype at a previously uncharacterized prion gene promoter location. Functional consequences of this genotype are currently being determined using a promoter luciferase assay. Non-prion genomic locations have also been identified as unique in abnormal and/or atypical BSE cases. These include the previously identified sites BTA6-97.6Mb (ANTXR2/BMP3), BTA10-21.4Mb (CHMP4A/
<figure><img src="image_77.png" /></figure>

## ABSTRACT

The possibility of on-going blood-borne transmission of variant Creutzfeldt-Jakob disease (vCJD) is a major concern for public health authorities, particularly in light of uncertainties surrounding the prevalence of subclinical vCJD infection in the human population. A robust and highly sensitive assay that can be applied to readily accessible samples (e.g. blood, urine), for detection of infected individuals before the onset of disease, would be of enormous benefit in preventing further transmission of vCJD. Using sheep experimentally infected with BSE as a model, we have previously demonstrated that the protein misfolding amplification assay (PMCA) can detect seeding activity in blood samples as early as 6 months post-infection. However, we also observed significant differences in incubation period, and transmission of infection by transfusion, associated with genetic variation at *PRNP* codon 141¹. The aim of this study was to determine the relationship between codon 141 PRNP genotype and the onset of detection of seeding activity in preclinical blood samples from orally-infected (donor) and intravenously-infected (recipient) sheep.

Samples were analysed using a microplate-based, miniaturized bead serial PMCA protocol²⁻³, with brain homogenate from tgShpXI transgenic mice expressing sheep ARQ PrP (with added dextran sulphate) as substrate, and undiluted buffy coat samples from BSE-infected and uninfect ed sheep as seed. In orally challenged sheep, blood samples were collected at intervals from 2 months post-infection until the onset of clinical signs. The first time point at which blood samples tested positive was very variable, ranging from 4 months to 12 months post-infection and there was no obvious correlation between codon 141 genotype and the onset of detection. In sheep infected by blood transfusion, the first preclinical blood samples were collected at 6 months post-infection, and majority of animals tested gave positive PMCA results at this time point, regardless of codon 141 *PRNP* genotype. For both orally and intravenously-infected sheep, all samples collected at time points following the onset of detection tested positive in the PMCA. The results demonstrate that the route of infection appears to have a much more significant influence than *PRNP* genotype on the onset of detection of seeding activity in preclinical blood samples. They also demonstrate that once seeding activity is detected in blood samples from infected individuals, it is consistently detected in samples from subsequent time points throughout the incubation period. This data provides valuable insights into the factors that may influence the sensitivity of detection of preclinical prion infection using blood-based assays.

## References

[1] Tan, et al. J Gen Virol. 2012;93(Pt 12): 2749-2756

[2] Moudjou, et al. MBio. 2013 31;5(1):e00829-13

[3] Lacroux, et al. PLoS Pathog. 2014;10:e1004202.

## 119. Host genetic factors impacting prion diseases: genomic analysis of phenotypically distinct bovine spongiform encephalopathy cattle

Sandor Dudas<sup>a,b</sup>, Graham Plastow<sup>c</sup>, James Cross<sup>b</sup> and Stefanie Czub<sup>a,b</sup>

<sup>a</sup>Canadian Food Inspection Agency, National Center for Animal Disease Lethbridge; <sup>b</sup>University of Calgary, Faculty of Veterinary Medicine; <sup>c</sup>University of Alberta, Agriculture, Food and Nutritional Science

CONTACT Sandor Dudas sandor.dudas@canada.ca

Populations and development of diagnostics and effective therapeutics for AD.

## ABSTRACT

**Background/Introduction:** Bovine spongiform encephalopathy (BSE)-challenged cattle have variable susceptibility, incubation times and in rare cases, even different disease phenotypes; our initial results suggest this may be related to host genetic factors. Attempts have been made to link bovine spongiform encephalopathy susceptibility with genotype using BSE field cases. These sample populations have a number of uncontrolled variables making analysis difficult and likely contribute to a lack of agreement between studies.

**Materials and Methods:** To identify genetic factors impacting BSE in a more controlled population, tissue samples from BSE-challenged cattle have been collected from the APHA (UK), the FLI (Germany), the USDA (USA), and the CFIA (Canada). DNA from these tissues are currently being genotyped to identify regions of the bovine genome which are associated with different BSE phenotypes. Once identified, targeted next generation sequencing will be used to find functionally relevant genetic changes. If important polymorphisms are found, gene-edited cell culture will be used to demonstrate the impacts these genetic variants have on prion expression and disease progression.

**Results:** Genotyping of short incubation BSE challenge cattle shows a significant link with the deletion genotype at a previously uncharacterized prion gene promoter location. Functional consequences of this genotype are currently being determined using a promoter luciferase assay. Non-prion genomic locations have also been identified as unique in abnormal and/or atypical BSE cases. These include the previously identified sites BTA6-97.6Mb (ANTXR2/BMP3), BTA10-21.4Mb (CHMP4A/
<figure><img src="image_78.png" /></figure>

MYH6), BTA20-4.90Mb (CART/STC2), and BTA20-39.85Mb (GDNF/SLC45A2) [1, 2]. We have also identified additional non-prion genomic regions unique to the atypical/abnormal BSE cattle genotyped so far. One example is a missense mutation at codon 439(G->S) in the phosphoserine rich protein encoded by the kiaa0232 gene. This amino acid (439G) is part of a conserved region in species naturally affected by prion diseases including sheep, goats and deer. The kiaa0232-encoded protein has also been identified as a binding partner of YHWAE/YHWAZ, members of the 14-3-3 protein family involved in signal transduction and apoptosis [3].

**Conclusions:** Genetic analysis of cattle with variable incubation or abnormal/atypical BSE could result in the identification of proteins/pathways involved in prion disease pathogenesis. Introducing these functionally relevant polymorphisms into cell culture models will hopefully result in changes in prion production/ accumulation providing evidence for an altered disease process. This will further our understanding of processes important in BSE progression and could provide future direction for selective breeding or therapeutics.

## References

[1] Murdoch, et al. BMC Genet. 2010;11:20.

[2] Thomson, et al. Prion. 2012;6(5):461-469.

[3] Bandyopadhyay, et al. Nature methods. 2010;7 (10):801-805.

# 120. Detection of CWD prion seeding activity in faeces demonstrates both consistent shedding and potential for environmental monitoring

Joanne M. Tennantª, Manci Liª, Davin M. Hendersonª, Nicholas J. Haleyᵇ, Candace K. Mathiasonª and Edward A. Hooverª

**CONTACT** Joanne M. Tennant joanne.tennant@rams.colostate.edu

## ABSTRACT

**Background:** Chronic wasting disease (CWD) is spreading in susceptible cervid populations in North America, Korea, and Europe. Environmental contamination and exposure to prions is considered to be a significant factor in horizontal CWD transmission. Currently disease surveillance is limited by the necessary presence and testability of infected animals. Previous studies have shown that excreta from CWD infected deer and elk contains prion

seeding activity. The detection of CWD through excreta, specifically faeces, could be beneficial in passive monitoring of CWD prevalence in endemic and emerging habitats.

**Methods:** Longitudinal collections of faeces from low-dose CWD inoculated deer were evaluated by real-time quaking induced conversion (RT-QuIC). To emulate common environmental weathering conditions, CWD-positive faecal samples were dried and exposed to UV light and prion seeding activity was evaluated by RT-QuIC before and after treatment. In addition, faeces from premises known to contain either CWD positive or negative cervids were collected and tested in RT-QuIC for prion seeding activity in a blinded experiment.

**Results:** CWD prion seeding activity was detected by RT-QuIC in faeces of five out of six low-dose CWD inoculated 96GG deer. The RT-QuIC detectable prion shedding in these deer corresponded with detection of $PrP^{CWD}$ by IHC in rectoanal mucosa-associated lymphoid tissue (RAMALT) biopsy and prion shedding was detected during both the pre-symptomatic and symptomatic stages of disease. Prion seeding activity was little affected by drying and exposure to ultraviolet light as both rectal collected and dried faeces showed prion seeding activity. Faeces collected from the pens containing CWD positive vs. negative deer correlated with presence vs. absence prion seeding activity, respectively.

**Conclusion:** These studies demonstrate that low-dose inoculated deer shed prion seeding activity in faeces through much of their disease course. Further, the prion seeding activity in faeces was still detectable after exposure to UV light and desiccation. These findings suggest that environmental monitoring of CWD via landscape faecal deposits may be possible as a means for monitoring CWD in populations of free-ranging deer and elk.

# 121. Tau prion strain propagation and cytotoxicity defined by live-cell imaging

Sang-Gyun Kangª,ᵇ, Ghazaleh Eskandari-Sedighiª,ᶜ, Nathalie Daudeª,ᵇ, Hristina Gapeshinaª, Jing Yangª and David Westawayª,ᵇ,ᶜ

**CONTACT** Sang-Gyun Kang sanggyun.kang@ualberta.ca

## ABSTRACT

**Introduction:** Tauopathies are a group of neurodegenerative disorders including Alzheimer's disease (AD),

<footnote>ªPrion Research Center, Department of Microbiology, Immunology, and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA;</footnote>

<footnote>ᵇDepartment of Microbiology and Pathology, Midwestern State University, Glendale, AZ, USA</footnote>

<footnote>ªCentre for Prions and Protein Folding Diseases; ᵇDivision of Neurology; ᵈDepartment of Biochemistry, University of Alberta, Edmonton, Canada</footnote>
<figure><img src="image_78.png" /></figure>

MYH6), BTA20-4.90Mb (CART/STC2), and BTA20-39.85Mb (GDNF/SLC45A2) [1, 2]. We have also identified additional non-prion genomic regions unique to the atypical/abnormal BSE cattle genotyped so far. One example is a missense mutation at codon 439(G->S) in the phosphoserine rich protein encoded by the kiaa0232 gene. This amino acid (439G) is part of a conserved region in species naturally affected by prion diseases including sheep, goats and deer. The kiaa0232-encoded protein has also been identified as a binding partner of YHWAE/YHWAZ, members of the 14-3-3 protein family involved in signal transduction and apoptosis [3].

**Conclusions:** Genetic analysis of cattle with variable incubation or abnormal/atypical BSE could result in the identification of proteins/pathways involved in prion disease pathogenesis. Introducing these functionally relevant polymorphisms into cell culture models will hopefully result in changes in prion production/ accumulation providing evidence for an altered disease process. This will further our understanding of processes important in BSE progression and could provide future direction for selective breeding or therapeutics.

## References

[1] Murdoch, et al. BMC Genet. 2010;11:20.

[2] Thomson, et al. Prion. 2012;6(5):461-469.

[3] Bandyopadhyay, et al. Nature methods. 2010;7 (10):801-805.

# 120. Detection of CWD prion seeding activity in faeces demonstrates both consistent shedding and potential for environmental monitoring

Joanne M. Tennantª, Manci Liª, Davin M. Hendersonª, Nicholas J. Haleyᵇ, Candace K. Mathiasonª and Edward A. Hooverª

ªPrion Research Center, Department of Microbiology, Immunology, and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA;

ᵇDepartment of Microbiology and Pathology, Midwestern State University, Glendale, AZ, USA

**CONTACT** Joanne M. Tennant joanne.tennant@rams.colostate.edu

## ABSTRACT

**Background:** Chronic wasting disease (CWD) is spreading in susceptible cervid populations in North America, Korea, and Europe. Environmental contamination and exposure to prions is considered to be a significant factor in horizontal CWD transmission. Currently disease surveillance is limited by the necessary presence and testability of infected animals. Previous studies have shown that excreta from CWD infected deer and elk contains prion

seeding activity. The detection of CWD through excreta, specifically faeces, could be beneficial in passive monitoring of CWD prevalence in endemic and emerging habitats.

**Methods:** Longitudinal collections of faeces from low-dose CWD inoculated deer were evaluated by real-time quaking induced conversion (RT-QuIC). To emulate common environmental weathering conditions, CWD-positive faecal samples were dried and exposed to UV light and prion seeding activity was evaluated by RT-QuIC before and after treatment. In addition, faeces from premises known to contain either CWD positive or negative cervids were collected and tested in RT-QuIC for prion seeding activity in a blinded experiment.

**Results:** CWD prion seeding activity was detected by RT-QuIC in faeces of five out of six low-dose CWD inoculated 96GG deer. The RT-QuIC detectable prion shedding in these deer corresponded with detection of $PrP^{CWD}$ by IHC in rectoanal mucosa-associated lymphoid tissue (RAMALT) biopsy and prion shedding was detected during both the pre-symptomatic and symptomatic stages of disease. Prion seeding activity was little affected by drying and exposure to ultraviolet light as both rectal collected and dried faeces showed prion seeding activity. Faeces collected from the pens containing CWD positive vs. negative deer correlated with presence vs. absence prion seeding activity, respectively.

**Conclusion:** These studies demonstrate that low-dose inoculated deer shed prion seeding activity in faeces through much of their disease course. Further, the prion seeding activity in faeces was still detectable after exposure to UV light and desiccation. These findings suggest that environmental monitoring of CWD via landscape faecal deposits may be possible as a means for monitoring CWD in populations of free-ranging deer and elk.

# 121. Tau prion strain propagation and cytotoxicity defined by live-cell imaging

Sang-Gyun Kangª,ᵇ, Ghazaleh Eskandari-Sedighiª,ᶜ, Nathalie Daudeª,ᵇ, Hristina Gapeshinaª, Jing Yangª and David Westawayª,ᵇ,ᶜ

ªCentre for Prions and Protein Folding Diseases; ᵇDivision of Neurology; ᵈDepartment of Biochemistry, University of Alberta, Edmonton, Canada

**CONTACT** Sang-Gyun Kang sanggyun.kang@ualberta.ca

## ABSTRACT

**Introduction:** Tauopathies are a group of neurodegenerative disorders including Alzheimer's disease (AD),
<figure><img src="image_79.png" /></figure>

progressive supranuclear palsy (PSP), corticobasal degenera-
tion (CBD), and frontotemporal dementia (FTD).
Tauopathies can be heterogeneous in clinical and patholo-
gical presentation and may be characterized by the intra-
cellular aggregation of microtubule-associated protein Tau
in neurofibrillary tangles (NFTs). Also, abnormally phos-
phorylated Tau becomes dissociated from neuronal micro-
tubules and accumulates in paired helical filaments (PHFs).
The conformational changes of monomeric soluble Tau
into oligomers and NFTs are thought to contribute to
neuronal dysfunction and eventually cell death. As prion-
like propagation of Tau ('strain' effects) has been invoked
to explain phenotypic diversity in Tau pathogenesis, we
have assessed this possibility using a cell culture system.

**Materials and Methods:** Previously, we reported dif-
ferent pathologic variations (classes 1–5) in low-expres-
ser TgTauP301L congenic mice encoding human Tau
(2N4R, Isoform Tau-F) [1]. Sarkosyl-insoluble Tau
fractions (hereafter, referred to as Tau fibrils) were
prepared from mouse brains of each pathology class.
HEK293 cells stably expressing a YFP-tagged human
Tau 4R repeat domain (RD) fragment that includes
aggregation-prone mutations (P301L/V337M, LM;
proaggregation) ('HEK-TauRD-LM-YFP cells') [2]
were transduced with liposome-protein complexes
derived from each class of Tau fibrils. Recombinant
Tau RD fibrils (recTauRD) were also assessed.
Transduced cells were split into six-well plates for the
measurement of insoluble Tau and for live-cell imaging
analysis.

**Results:** Each class of Tau fibrils (including recTauRD) could show heterogeneous morphologies for Tau inclusions in the HEK293-TauRD-LM-YFP cells. Nonetheless, we observed three putative Tau prion strains from brain samples as defined by striking differences in their subcellular distribution of Tau deposits; a large mass of aggregated Tau with no specific pattern (amorphous: ES1), juxtanuclear and nuclear membrane inclusions (juxtanuclear: ES2), and granular nuclear inclusions (speckles: ES3). Live-cell imaging analysis revealed that ES1 and ES2 forms were toxic to the cells, whereas cells with ES3-type inclusions showed no cell death over a period of 16 h. Interestingly, nuclear blebs were prominent and co-localized with ES1 and ES2 Tau fluorescent signals, indicating Tau-induced nuclear damage.

**Conclusions:** TgTauP301L mice, an inbred congenic model of genetic Tauopathy, generate more than one form of biologically active Tau, as revealed by differing patterns of propagation and cytotoxicity in the cell-based assays. We also conclude that nuclear deformation contributes to neurotoxicity in a sub-set of Tauopathies.

References

[1] Eskandari-Sedighi, Daude, et al. Mol Neurodegener. 4 October 2017;12(1):72.

[2] Kaufman, Sanders, et al. Neuron. 23 November 2016;92(4):796–812.

122. Diagnosis and biomarkers of Parkinson's and Dementia with Lewy bodies: rapid and ultra-sensitive quantitation of disease-associated α-synuclein by αSyn RT-QuIC

Christina D. Orrùª, Bradley R Grovemanª, Andrew G
Hughsonª, Lynne D Raymondª, Gianluigi Zanussoᵇ,
Bernardino Ghettiᶜ, Katrina J Campbellª, Natália do
Carmo Ferreiraª, Jiri Safarᵈ, Douglas Galaskoᵉ, Oskar
Hanssonf, g and Byron Caugheyª

ªLaboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT, USA; ᵇDepartment of Neurosciences, Biomedicine and Movement Sciences, University of Verona, Verona, Italy; ᶜIndiana University School of Medicine, Indianapolis, IN, USA; ᵈDepartment of Pathology, Case Western Reserve University School of Medicine, Cleveland, OH, USA; ᵉDepartment of Neurosciences, University of California-San Diego, La Jolla, CA, USA; ᵈClinical Memory Research Unit, Department of Clinical Sciences Malmö, Lund University, Lund, Sweden; ᵉMemory Clinic, Skåne University Hospital, Malmö, Sweden

CONTACT Christina D. Orrù christina.orru@nih.gov

ABSTRACT

Diagnosis of synucleinopathies such as Parkinson disease and dementia with Lewy bodies can be aided by assays for the detection and quantification of pathogenic disease-associated forms of α-synuclein (αSynD). These assays need to be sufficiently sensitive, specific, and practical for analysis of accessible diagnostic samples. Two recent αSynD seed amplification assays have provided valuable prototypes for detection of αSynD in patients’ cerebrospinal fluid (CSF). However, these assays require 5–13 days to perform. We have developed an improved α-synuclein real time quaking-induced conversion (αSyn RT-QuIC) assay with similar sensitivity and specificity that provides quantitative results in 1–2 days. Our blinded analysis of cerebrospinal fluid from 29 synucleinopathy cases (12 Parkinson’s and 17 dementia with Lewy bodies) and 31 non-synucleinopathy controls, including 16 Alzheimer’s cases, yielded 93% diagnostic sensitivity and 100% specificity. End-point dilution analyses detected αSynD in as little as 0.2 µL of CSF. Furthermore, our test allowed us to quantitate relative amounts of αSynD in CSF samples. We are currently testing additional well-characterized blinded panels of CSF samples from individuals diagnosed with
<figure><img src="image_79.png" /></figure>

progressive supranuclear palsy (PSP), corticobasal degenera-
tion (CBD), and frontotemporal dementia (FTD).
Tauopathies can be heterogeneous in clinical and patholo-
gical presentation and may be characterized by the intra-
cellular aggregation of microtubule-associated protein Tau
in neurofibrillary tangles (NFTs). Also, abnormally phos-
phorylated Tau becomes dissociated from neuronal micro-
tubules and accumulates in paired helical filaments (PHFs).
The conformational changes of monomeric soluble Tau
into oligomers and NFTs are thought to contribute to
neuronal dysfunction and eventually cell death. As prion-
like propagation of Tau ('strain' effects) has been invoked
to explain phenotypic diversity in Tau pathogenesis, we
have assessed this possibility using a cell culture system.

**Materials and Methods:** Previously, we reported dif-
ferent pathologic variations (classes 1–5) in low-expres-
ser TgTauP301L congenic mice encoding human Tau
(2N4R, Isoform Tau-F) [1]. Sarkosyl-insoluble Tau
fractions (hereafter, referred to as Tau fibrils) were
prepared from mouse brains of each pathology class.
HEK293 cells stably expressing a YFP-tagged human
Tau 4R repeat domain (RD) fragment that includes
aggregation-prone mutations (P301L/V337M, LM;
proaggregation) ('HEK-TauRD-LM-YFP cells') [2]
were transduced with liposome-protein complexes
derived from each class of Tau fibrils. Recombinant
Tau RD fibrils (recTauRD) were also assessed.
Transduced cells were split into six-well plates for the
measurement of insoluble Tau and for live-cell imaging
analysis.

**Results:** Each class of Tau fibrils (including recTauRD) could show heterogeneous morphologies for Tau inclusions in the HEK293-TauRD-LM-YFP cells. Nonetheless, we observed three putative Tau prion strains from brain samples as defined by striking differences in their subcellular distribution of Tau deposits; a large mass of aggregated Tau with no specific pattern (amorphous: ES1), juxtanuclear and nuclear membrane inclusions (juxtanuclear: ES2), and granular nuclear inclusions (speckles: ES3). Live-cell imaging analysis revealed that ES1 and ES2 forms were toxic to the cells, whereas cells with ES3-type inclusions showed no cell death over a period of 16 h. Interestingly, nuclear blebs were prominent and co-localized with ES1 and ES2 Tau fluorescent signals, indicating Tau-induced nuclear damage.

**Conclusions:** TgTauP301L mice, an inbred congenic model of genetic Tauopathy, generate more than one form of biologically active Tau, as revealed by differing patterns of propagation and cytotoxicity in the cell-based assays. We also conclude that nuclear deformation contributes to neurotoxicity in a sub-set of Tauopathies.

**References**

[1] Eskandari-Sedighi, Daude, et al. Mol Neurodegener. 4 October 2017;12(1):72.

[2] Kaufman, Sanders, et al. Neuron. 23 November 2016;92(4):796-812.

122. Diagnosis and biomarkers of Parkinson's and Dementia with Lewy bodies: rapid and ultra-sensitive quantitation of disease-associated α-synuclein by αSyn RT-QuIC

Christina D. Orrùª, Bradley R Grovemanª, Andrew G
Hughsonª, Lynne D Raymondª, Gianluigi Zanussoᵇ,
Bernardino Ghettiᶜ, Katrina J Campbellª, Natália do
Carmo Ferreiraª, Jiri Safarᵈ, Douglas Galaskoᵉ, Oskar
Hanssonf, g and Byron Caugheyª

ªLaboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT, USA; ᵇDepartment of Neurosciences, Biomedicine and Movement Sciences, University of Verona, Verona, Italy; ᶜIndiana University School of Medicine, Indianapolis, IN, USA; ᵈDepartment of Pathology, Case Western Reserve University School of Medicine, Cleveland, OH, USA; ᵉDepartment of Neurosciences, University of California-San Diego, La Jolla, CA, USA; ᵈClinical Memory Research Unit, Department of Clinical Sciences Malmö, Lund University, Lund, Sweden; ᵉMemory Clinic, Skåne University Hospital, Malmö, Sweden

CONTACT Christina D. Orrù christina.orru@nih.gov

ABSTRACT

Diagnosis of synucleinopathies such as Parkinson disease and dementia with Lewy bodies can be aided by assays for the detection and quantification of pathogenic disease-associated forms of α-synuclein (αSynD). These assays need to be sufficiently sensitive, specific, and practical for analysis of accessible diagnostic samples. Two recent αSynD seed amplification assays have provided valuable prototypes for detection of αSynD in patients’ cerebrospinal fluid (CSF). However, these assays require 5–13 days to perform. We have developed an improved α-synuclein real time quaking-induced conversion (αSyn RT-QuIC) assay with similar sensitivity and specificity that provides quantitative results in 1–2 days. Our blinded analysis of cerebrospinal fluid from 29 synucleinopathy cases (12 Parkinson’s and 17 dementia with Lewy bodies) and 31 non-synucleinopathy controls, including 16 Alzheimer’s cases, yielded 93% diagnostic sensitivity and 100% specificity. End-point dilution analyses detected αSynD in as little as 0.2 µL of CSF. Furthermore, our test allowed us to quantitate relative amounts of αSynD in CSF samples. We are currently testing additional well-characterized blinded panels of CSF samples from individuals diagnosed with
<figure><img src="image_80.png" /></figure>

synucleinopathies and controls. Moreover, we have also
developed a capture protocol for the detection of αSyn<sup>D</sup>
in human plasma spiked with αSyn positive brain
homogenate and in endogenous CSF samples from
individuals affected by synucleinopathies. We can now
isolate αSyn<sup>D</sup> from several biological samples for sensi-
tive and specific detection by αSyn RT-QuIC.

**123. Detection of prion seeds throughout the eyes of sporadic Creutzfeldt-Jakob disease patients**

Christina Orruª, Katrin Soldauᵇ, Christian Cordanoᶜ, Jorge Llibre-Guerraᵈ, Ari J. Greenᵉ, Henry Sanchezᶜ, Bradley R. Grovemanª, Steven D. Edlandf,g, Jiri G. Safarh,i, Jonathan H. Linb, Byron Caugheyª, Michael D. Geschwindc and Christina J. Sigurdsonb,j

ªLaboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Hamilton, MT, USA; ᵇDepartment of Pathology, University of California, San Diego, La Jolla, CA USA; ᶜDepartment of Neurology, Memory and Aging Center, University of California, San Francisco (UCSF), San Francisco, CA, USA; ᵈCognitive and Behavioral Research Unit, National Institute of Neurology, Havana, Cuba; ᵉDepartment of Neurology, Multiple Sclerosis Center, University of California, San Francisco (UCSF), San Francisco, CA, USA; ᵈDepartments of Family Medicine & Public Health and ᵉNeurosciences, University of California, San Diego, La Jolla, CA USA; ᵈDepartments of Pathology and ᵈNeurology, Case Western Reserve University, Cleveland, OH, USA; ᵈDepartment of Pathology, Immunology, and Microbiology, University of California, Davis, Davis, CA, USA

CONTACT Christina Orru christina.orru@nih.gov

Orrù and Soldau, mBIO 2018 9:e02095-18. https://doi.
org/10.1128/ mBio.02095-18

ABSTRACT

Cases of iatrogenic Creutzfeldt-Jakob disease (CJD) have
been reported from corneal transplants, yet the levels of
prions in the eye remain unknown. Approximately, 40%
of sporadic CJD patients develop visual symptoms and
often seek ophthalmological consultation. This study
probed the occurrence, level, and distribution of prions
in the eyes of patients with sCJD, the most common
prion disease in humans. We used the highly sensitive
real time quaking-induced conversion (RT-QuIC) assay
to measure post-mortem levels of prion seeding activity
in cornea, lens, ocular fluid, retina, choroid, sclera, optic
nerve, and extraocular muscle. This is the largest series
of sCJD patient eyes studied by any assay to date. We
detected prion seeding activity in 100% of sCJD eyes
representing three common sCJD subtypes with a four
log-fold range of seeding activity observed across indi-
viduals. The retina consistently showed the highest prion

seeding activity. Within the retina, prion deposits were
also detected by immunohistochemistry in the outer
plexiform layer, and in some eyes the inner plexiform
layer. With increased distance from the brain prion seed
levels by RT-QuIC declined in eye tissues. Collectively,
these results reveal that sCJD patients accumulate prions
throughout the eyes, indicating the potential diagnostic
utility as well as the possible biohazard.

124. Longitudinal studies of CWD after low-dose oral exposure in white-tailed deer

Clare E. Hoover*, Nathaniel D. Denkers, Kristen A. Davenport, Davin M. Henderson, Amy V. Nalls, Erin McNulty, Joanne Tennant, Sarah Cooper, Manci Li, Lauren Bracchi, Candace K. Mathiason and Edward A. Hoover

Prion Research Center, Department of Microbiology, Immunology, and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA

CONTACT Edward A. Hoover edward.hoover@colostate.edu

*Present address: AstraZeneca, Waltham, NJ

ABSTRACT

**Background:** The facile transmission of chronic wasting disease (CWD) in North America, Asia and Europe continues despite exposure to very low concentrations of prions shed by infected cervids in secreta and excreta. Explanations for this enigma could be that excreted prions may have enhanced infectivity and/or that the infectious prion dose is just quite low. Historical studies exploring CWD pathogenesis in deer have used exposures to CWD-positive brain homogenates containing from 0.5 to 10 g of brain – doses much higher than those measured in excreta, and thereby likely to ever occur in nature. We thereby sought to more closely emulate natural exposure context in our current experimental studies.

**Methods:** To explore how the origin or infectious dose of CWD prions may influence disease transmission or pathogenesis, we orally exposed cohorts of *n* = 4/group white-tailed deer to low doses of CWD presented as either CWD-positive brain homogenate (containing either 1 mg or 300 ng of brain) or to an amount of pooled saliva from CWD+ donors and containing prion seeding activity (by RT-QuIC) equivalent to 300 ng of CWD+ brain [1]. Inoculated deer were then longitudinally monitored for: (a) onset of prion infection in biopsied tonsil and recto-anal lymphoid tissues by RT-QuIC and immunohisto-chemistry; (b) prion shedding in saliva and faeces (by
<figure><img src="image_80.png" /></figure>

synucleinopathies and controls. Moreover, we have also
developed a capture protocol for the detection of αSyn<sup>D</sup>
in human plasma spiked with αSyn positive brain
homogenate and in endogenous CSF samples from
individuals affected by synucleinopathies. We can now
isolate αSyn<sup>D</sup> from several biological samples for sensi-
tive and specific detection by αSyn RT-QuIC.

**123. Detection of prion seeds throughout the eyes of sporadic Creutzfeldt-Jakob disease patients**

Christina Orruª, Katrin Soldauᵇ, Christian Cordanoᶜ,
Jorge Llibre-Guerraᵈ, Ari J. Greenᵉ, Henry Sanchezᶜ,
Bradley R. Grovemanª, Steven D. Edlandf,g, Jiri G.
Safarh,i, Jonathan H. Linb, Byron Caugheyª, Michael
D. Geschwindc and Christina J. Sigurdsonb,j

ªLaboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Hamilton, MT, USA; ᵇDepartment of Pathology, University of California, San Diego, La Jolla, CA USA; ᶜDepartment of Neurology, Memory and Aging Center, University of California, San Francisco (UCSF), San Francisco, CA, USA; ᵈCognitive and Behavioral Research Unit, National Institute of Neurology, Havana, Cuba; ᵉDepartment of Neurology, Multiple Sclerosis Center, University of California, San Francisco (UCSF), San Francisco, CA, USA; ᵈDepartments of Family Medicine & Public Health and ᵉNeurosciences, University of California, San Diego, La Jolla, CA USA; ᵈDepartments of Pathology and ᵈNeurology, Case Western Reserve University, Cleveland, OH, USA; ᵈDepartment of Pathology, Immunology, and Microbiology, University of California, Davis, Davis, CA, USA

CONTACT Christina Orru christina.orru@nih.gov

Orrù and Soldau, mBIO 2018 9:e02095-18. https://doi.
org/10.1128/ mBio.02095-18

ABSTRACT

Cases of iatrogenic Creutzfeldt-Jakob disease (CJD) have
been reported from corneal transplants, yet the levels of
prions in the eye remain unknown. Approximately, 40%
of sporadic CJD patients develop visual symptoms and
often seek ophthalmological consultation. This study
probed the occurrence, level, and distribution of prions
in the eyes of patients with sCJD, the most common
prion disease in humans. We used the highly sensitive
real time quaking-induced conversion (RT-QuIC) assay
to measure post-mortem levels of prion seeding activity
in cornea, lens, ocular fluid, retina, choroid, sclera, optic
nerve, and extraocular muscle. This is the largest series
of sCJD patient eyes studied by any assay to date. We
detected prion seeding activity in 100% of sCJD eyes
representing three common sCJD subtypes with a four
log-fold range of seeding activity observed across indi-
viduals. The retina consistently showed the highest prion

seeding activity. Within the retina, prion deposits were
also detected by immunohistochemistry in the outer
plexiform layer, and in some eyes the inner plexiform
layer. With increased distance from the brain prion seed
levels by RT-QuIC declined in eye tissues. Collectively,
these results reveal that sCJD patients accumulate prions
throughout the eyes, indicating the potential diagnostic
utility as well as the possible biohazard.

124. Longitudinal studies of CWD after low-dose oral exposure in white-tailed deer

Clare E. Hoover*, Nathaniel D. Denkers, Kristen A.
Davenport, Davin M. Henderson, Amy V. Nalls, Erin
McNulty, Joanne Tennant, Sarah Cooper, Manci Li,
Lauren Bracchi, Candace K. Mathiason and Edward
A. Hoover

Prion Research Center, Department of Microbiology, Immunology, and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA

CONTACT Edward A. Hoover edward.hoover@colostate.edu

*Present address: AstraZeneca, Waltham, NJ

ABSTRACT

**Background:** The facile transmission of chronic wasting disease (CWD) in North America, Asia and Europe continues despite exposure to very low concentrations of prions shed by infected cervids in secreta and excreta. Explanations for this enigma could be that excreted prions may have enhanced infectivity and/or that the infectious prion dose is just quite low. Historical studies exploring CWD pathogenesis in deer have used exposures to CWD-positive brain homogenates containing from 0.5 to 10 g of brain – doses much higher than those measured in excreta, and thereby likely to ever occur in nature. We thereby sought to more closely emulate natural exposure context in our current experimental studies.

**Methods:** To explore how the origin or infectious dose of CWD prions may influence disease transmission or pathogenesis, we orally exposed cohorts of *n* = 4/group white-tailed deer to low doses of CWD presented as either CWD-positive brain homogenate (containing either 1 mg or 300 ng of brain) or to an amount of pooled saliva from CWD+ donors and containing prion seeding activity (by RT-QuIC) equivalent to 300 ng of CWD+ brain [1]. Inoculated deer were then longitudinally monitored for: (a) onset of prion infection in biopsied tonsil and recto-anal lymphoid tissues by RT-QuIC and immunohisto-chemistry; (b) prion shedding in saliva and faeces (by
<figure><img src="image_81.png" /></figure>

RT-QuIC); (c) onset of clinical signs (weekly observation and scoring); and (d) time to clinical disease.

**Results:** We first detected CWD infection by RT-QuIC seeding activity in tonsil biopsies at 6 or 9 months post exposure, in all inoculation cohorts. Prion shedding in saliva was detected (by RT-QuIC or PMCA-RT-QuIC [2]) concurrent with the first positive tonsil biopsy. Detection of prion seeding activity in faeces was less common overall and its onset observed after positivity in recto-anal lymphoid tissue biopsies. In comparison to historical studies, in which >1,000-fold higher inoculation doses were used, the time to first detection on infection (by tonsil biopsy) was prolonged by ~6 months. However, among the low-dose inoculation cohorts in this study, neither the attack rate, prion shedding profile, nor long-term disease course differed significantly, including the deer exposed to prions of saliva vs. brain origin.

**Conclusions:** These studies demonstrate that (a) much lower doses of CWD prions that have been used historically for point source exposure studies are sufficient to induce prion infection, shedding, and disease; and (b) although time to first detectable positivity is lengthened, the qualitative aspects of disease pathogenesis thereafter are indistinguishable by inoculum source or exposure magnitude.

Our future studies will aim at establishing the minimum oral infectious dose for CWD in deer and on exploring exposure cofactors.

## References

[1] Hoover CE, et al. in preparation, 2019

[2] Davenport KA, et al. J Clin Microbiol. 27 August 2018;56(9).

Supported by NIH R01-NS-061902, P01-AI-077774, F30-ODO-118,143, T32-OD0-10,437

## 125. Bioassay confirms the relevance of surf-PMCA for human prion decontamination studies

Maxime Bélondradeª, Simon Nicotª, Christelle Jasª,ᵇ, Vincent Béringueᵇ, Chantal Fournier-Wirthª, Sylvain Lehmannᶜ and Daisy Bougardª*

CONTACT Daisy Bougard daisy.bougard@efs.sante.fr

Abstract not available.

## 126. Dickinson, the forgotten man of the prion story

James Hopeª and Jason Bartzᵇ

ªOne Health, Norwich, Norfolk, UK; ᵇCreighton University, Omaha, Nebraska, USA

CONTACT James Hope jameshope8@outlook.com

<figure><img src="image_82.png" /></figure>

Alan Dickinson, geneticist, born 30 March 1930; died 27 September 2017

Alan Dickinson died just after the Prion 2017 meeting in Edinburgh [1] and, sadly, his iconoclastic stance (on everything) and working tenet of the importance of being awkward, has meant his contribution to our prion world has faded much too quickly. To redress the balance a wee bit, let's be clear: he was a fantastic geneticist and, with Hugh Fraser and Moira Bruce, sorted out much of the confusion and mystery of scrapie in sheep that raged in the late 1950s and 1960s. His post-graduate training in genetics was meticulously applied to the breeding of mice and sheep, and he was able to biologically define alleles of a genetic locus (Sinc in mice) in both which appear to control the timing and tempo of the inoculated (and natural) disease with clockwork precision. The methods of strain typing using vacuolar pathology and mice of defined prion protein genotype (PrP is a product of the Sinc locus) are still used today in prion and prion-like disease research, and strain theory in TSEs and other neurodegenerative disease continues to intrigue and test our understanding of mechanisms of propagation, blocking and phenotype stability. It is interesting to speculate how much of his 'awkward' legacy has been explained by prions. What is our molecular explanation for breakdown of the 87A strain to ME7? How does one slow replicating strain prevent disease with the characteristics of a later-inoculated, short-incubation period prion? Current work does indicate that prion strains compete for PrPᶜ in a common

<footnote>ªPathogenesis and control of chronic infections, Etablissement Français du Sang, Inserm, Université de Montpellier, Montpellier, France; ᵇInstitut National de la Recherche Agronomique (INRA), UR892, Virologie Immunologie Moléculaires, Jouy-en-Josas, France; ᶜCHRU de Montpellier and Université de Montpellier, IRMB, INSERM U1183, Laboratoire de Biochimie Protéomique Clinique, Montpellier, France</footnote>
<figure><img src="image_81.png" /></figure>

RT-QuIC); (c) onset of clinical signs (weekly observation and scoring); and (d) time to clinical disease.

**Results:** We first detected CWD infection by RT-QuIC seeding activity in tonsil biopsies at 6 or 9 months post exposure, in all inoculation cohorts. Prion shedding in saliva was detected (by RT-QuIC or PMCA-RT-QuIC [2]) concurrent with the first positive tonsil biopsy. Detection of prion seeding activity in faeces was less common overall and its onset observed after positivity in recto-anal lymphoid tissue biopsies. In comparison to historical studies, in which >1,000-fold higher inoculation doses were used, the time to first detection on infection (by tonsil biopsy) was prolonged by ~6 months. However, among the low-dose inoculation cohorts in this study, neither the attack rate, prion shedding profile, nor long-term disease course differed significantly, including the deer exposed to prions of saliva vs. brain origin.

**Conclusions:** These studies demonstrate that (a) much lower doses of CWD prions that have been used historically for point source exposure studies are sufficient to induce prion infection, shedding, and disease; and (b) although time to first detectable positivity is lengthened, the qualitative aspects of disease pathogenesis thereafter are indistinguishable by inoculum source or exposure magnitude.

Our future studies will aim at establishing the minimum oral infectious dose for CWD in deer and on exploring exposure cofactors.

## References

[1] Hoover CE, et al. in preparation, 2019

[2] Davenport KA, et al. J Clin Microbiol. 27 August 2018;56(9).

Supported by NIH R01-NS-061902, P01-AI-077774, F30-ODO-118,143, T32-OD0-10,437

## 125. Bioassay confirms the relevance of surf-PMCA for human prion decontamination studies

Maxime Bélondradeª, Simon Nicotª, Christelle Jasª,ᵇ, Vincent Béringueᵇ, Chantal Fournier-Wirthª, Sylvain Lehmannᶜ and Daisy Bougardª*

CONTACT Daisy Bougard daisy.bougard@efs.sante.fr

Abstract not available.

## 126. Dickinson, the forgotten man of the prion story

James Hopeª and Jason Bartzᵇ

ªOne Health, Norwich, Norfolk, UK; ᵇCreighton University, Omaha, Nebraska, USA

CONTACT James Hope jameshope8@outlook.com

<figure><img src="image_82.png" /></figure>

Alan Dickinson, geneticist, born 30 March 1930; died 27 September 2017

Alan Dickinson died just after the Prion 2017 meeting in Edinburgh [1] and, sadly, his iconoclastic stance (on everything) and working tenet of the importance of being awkward, has meant his contribution to our prion world has faded much too quickly. To redress the balance a wee bit, let's be clear: he was a fantastic geneticist and, with Hugh Fraser and Moira Bruce, sorted out much of the confusion and mystery of scrapie in sheep that raged in the late 1950s and 1960s. His post-graduate training in genetics was meticulously applied to the breeding of mice and sheep, and he was able to biologically define alleles of a genetic locus (Sinc in mice) in both which appear to control the timing and tempo of the inoculated (and natural) disease with clockwork precision. The methods of strain typing using vacuolar pathology and mice of defined prion protein genotype (PrP is a product of the Sinc locus) are still used today in prion and prion-like disease research, and strain theory in TSEs and other neurodegenerative disease continues to intrigue and test our understanding of mechanisms of propagation, blocking and phenotype stability. It is interesting to speculate how much of his 'awkward' legacy has been explained by prions. What is our molecular explanation for breakdown of the 87A strain to ME7? How does one slow replicating strain prevent disease with the characteristics of a later-inoculated, short-incubation period prion? Current work does indicate that prion strains compete for PrPᶜ in a common

<footnote>ªPathogenesis and control of chronic infections, Etablissement Français du Sang, Inserm, Université de Montpellier, Montpellier, France; ᵇInstitut National de la Recherche Agronomique (INRA), UR892, Virologie Immunologie Moléculaires, Jouy-en-Josas, France; ᶜCHRU de Montpellier and Université de Montpellier, IRMB, INSERM U1183, Laboratoire de Biochimie Protéomique Clinique, Montpellier, France</footnote>
<figure><img src="image_83.png" /></figure>

population of cells [2], consistent with AGD's original 'replication site' hypothesis [3]. And 'strain-specific' co-factors, the prion's host-independent genome, are still sought after today, but remain elusive [4]. Crucially, he did use his insights for practical good, *inter alia* predicting the dangers of human growth hormone (hGH) therapy for pituitary dwarfism, and validating methods for its safe purification [5], decades before the current controversy of hGH therapy, 'Alzheimer' prions and Aβ pathology. He died continuing to search for an elusive second molecule to explain these biological phenomena – the iconoclast in him never accepted PrP (or the nomenclature) as the sole component of a naturally-infected prion – and he ruffled a few feathers in old age by continually defining molecular biology as 'fishing without a licence', rephrasing Chargaff's view of it as 'the practice of biochemistry without a licence' [6]. Even so, his life's work deserves recognition to this day in the bibliographies of our publications.

**KEYWORDS:** virino; strains; *Sinc*; mouse; genetics; vacuolation; stability classes; hGH; history of science

## References

[1] Green, E. Guardian (Manchester, UK) 23 November 2017. Geneticist who carried out groundbreaking research into the behaviour of diseases including scrapie and CJD [cited 15 February 2018].

[2] Eckland TE, Shikiya RA, Bartz JC. Independent amplification of co-infected long incubation period low conversion efficiency prion strains. PLoS Pathog. 2018;14:e1007323.

[3] Dickinson, AG, Outram GW. The scrapie replication-site hypothesis and its implications for pathogenesis. In: Prusiner SB, Hadlow WJ, editor, Slow transmissible diseases of the central nervous system. Vol. 2. New York, NY: Academic Press, Inc.; 1979. p. 13–31.

[4] Saá P, Sferrazza GF, Ottenberg G, et al. Strain-specific role of RNAs in prion replication. J Virol. 2012;86:10494–10504.

[5] Taylor DM, et al. Preparation of growth hormone free from contamination with unconventional slow viruses. Lancet. 1985 Aug 3; 8449:260–262.

[6] Wright, P. Guardian (Manchester, UK) 2 July 2002. Disillusioned biochemist who pioneered our understanding of DNA [cited 7 February 2019].

**127. Focal and diverse deposition in a mouse model of 4-repeat tauopathy is paralleled by distinct biochemical and cellular signatures; close relationship to Tau species in P301L patients**

D. Westaway, N. Daude, G. Eskandari-Sedighi, S. Kang, C. Kim, S. Fleck, J. Yang, H. Wille, E. Gelpi and J. G. Safar

## ABSTRACT

**Background:** Mutations in the Tau (MAPT) gene can cause neurodegenerative diseases such as frontotemporal lobar degeneration (FTLD) but, strikingly, patients with the same mutation have very different clinical phenotypes; the cause of this diversity is not understood. Heterogeneities have also been observed in a transgenic (Tg) mouse line expressing low levels of mutant human P301L Tau and backcrosses of founder stocks of mice to inbred genetic backgrounds to attenuate contributions from modifier loci failed to eliminate variability in disease-related phenotypes (Eskandari-Sedighi et al., 2017). Here biochemical and cellular assays have been used to assess chemical and biological characteristics of P301L Tau species from Tg.P301L mice and from human FTLD.

**Methods:** We used Tg.P301L mice from C57BL/6Tac, 129/SvEvTac and FVB/NJ genetic backgrounds and brain material from eight Iberian FTLD patients with the same P301L mutation and similar MAPT locus haplotypes (Borrego-Ecija et al., 2017). Tau species were profiled using microfluidic western assays, immunostaining, conformation-dependent immunoassays (CDI), conformational stability assays (CSAs), trypsin digestion, as well as by kinetic and morphological performance measures in a cell-based seeding assay. Electron microscopy (EM) was used to investigate fibril morphology.

**Results:** TgP301L mice yielded six patterns of Tau deposition in neurons and astrocytes; these included brain stem and focal deposition and in Class VI, present in half of aged mice, tangle-like deposits in the *locus coeruleus*. As defined by biochemical assays, three distinct Tau signatures were present in the brains of inbred Tg mice, these signatures often correlating with pathology classes. EM revealed that brains of Classes I, II, III and IV animals exhibit at least three distinguishable fibril morphology; the most abundant being straight filaments with twisted ribbon-like filament and coiled filaments being present in some classes. Analyses of detergent-insoluble tau in the frontal cortex of FTLD patients demonstrated remarkable conformational heterogeneity with at least two Tau signatures that mimicked a CSA pattern found in a subset of aged TgP301L mice. Toxicity above baseline in transfected cells was greatest with brain-derived material, with different preparations differing in their kinetics of cell loss and fluorescent signals at the nuclear margin.

**Conclusions:** Heterogeneities in tau pathology and chemical signatures were observed within congenic stocks of TgP301L mice and in different P301L patients; crucially, signatures present in patients also manifested in
<figure><img src="image_83.png" /></figure>

population of cells [2], consistent with AGD's original 'replication site' hypothesis [3]. And 'strain-specific' co-factors, the prion's host-independent genome, are still sought after today, but remain elusive [4]. Crucially, he did use his insights for practical good, *inter alia* predicting the dangers of human growth hormone (hGH) therapy for pituitary dwarfism, and validating methods for its safe purification [5], decades before the current controversy of hGH therapy, 'Alzheimer' prions and Aβ pathology. He died continuing to search for an elusive second molecule to explain these biological phenomena – the iconoclast in him never accepted PrP (or the nomenclature) as the sole component of a naturally-infected prion – and he ruffled a few feathers in old age by continually defining molecular biology as 'fishing without a licence', rephrasing Chargaff's view of it as 'the practice of biochemistry without a licence' [6]. Even so, his life's work deserves recognition to this day in the bibliographies of our publications.

**KEYWORDS:** virino; strains; *Sinc*; mouse; genetics; vacuolation; stability classes; hGH; history of science

## References

[1] Green, E. Guardian (Manchester, UK) 23 November 2017. Geneticist who carried out groundbreaking research into the behaviour of diseases including scrapie and CJD [cited 15 February 2018].

[2] Eckland TE, Shikiya RA, Bartz JC. Independent amplification of co-infected long incubation period low conversion efficiency prion strains. PLoS Pathog. 2018;14:e1007323.

[3] Dickinson, AG, Outram GW. The scrapie replication-site hypothesis and its implications for pathogenesis. In: Prusiner SB, Hadlow WJ, editor, Slow transmissible diseases of the central nervous system. Vol. 2. New York, NY: Academic Press, Inc.; 1979. p. 13–31.

[4] Saá P, Sferrazza GF, Ottenberg G, et al. Strain-specific role of RNAs in prion replication. J Virol. 2012;86:10494–10504.

[5] Taylor DM, et al. Preparation of growth hormone free from contamination with unconventional slow viruses. Lancet. 1985 Aug 3; 8449:260–262.

[6] Wright, P. Guardian (Manchester, UK) 2 July 2002. Disillusioned biochemist who pioneered our understanding of DNA [cited 7 February 2019].

**127. Focal and diverse deposition in a mouse model of 4-repeat tauopathy is paralleled by distinct biochemical and cellular signatures; close relationship to Tau species in P301L patients**

D. Westaway, N. Daude, G. Eskandari-Sedighi, S. Kang, C. Kim, S. Fleck, J. Yang, H. Wille, E. Gelpi and J. G. Safar

## ABSTRACT

**Background:** Mutations in the Tau (MAPT) gene can cause neurodegenerative diseases such as frontotemporal lobar degeneration (FTLD) but, strikingly, patients with the same mutation have very different clinical phenotypes; the cause of this diversity is not understood. Heterogeneities have also been observed in a transgenic (Tg) mouse line expressing low levels of mutant human P301L Tau and backcrosses of founder stocks of mice to inbred genetic backgrounds to attenuate contributions from modifier loci failed to eliminate variability in disease-related phenotypes (Eskandari-Sedighi et al., 2017). Here biochemical and cellular assays have been used to assess chemical and biological characteristics of P301L Tau species from Tg.P301L mice and from human FTLD.

**Methods:** We used Tg.P301L mice from C57BL/6Tac, 129/SvEvTac and FVB/NJ genetic backgrounds and brain material from eight Iberian FTLD patients with the same P301L mutation and similar MAPT locus haplotypes (Borrego-Ecija et al., 2017). Tau species were profiled using microfluidic western assays, immunostaining, conformation-dependent immunoassays (CDI), conformational stability assays (CSAs), trypsin digestion, as well as by kinetic and morphological performance measures in a cell-based seeding assay. Electron microscopy (EM) was used to investigate fibril morphology.

**Results:** TgP301L mice yielded six patterns of Tau deposition in neurons and astrocytes; these included brain stem and focal deposition and in Class VI, present in half of aged mice, tangle-like deposits in the *locus coeruleus*. As defined by biochemical assays, three distinct Tau signatures were present in the brains of inbred Tg mice, these signatures often correlating with pathology classes. EM revealed that brains of Classes I, II, III and IV animals exhibit at least three distinguishable fibril morphology; the most abundant being straight filaments with twisted ribbon-like filament and coiled filaments being present in some classes. Analyses of detergent-insoluble tau in the frontal cortex of FTLD patients demonstrated remarkable conformational heterogeneity with at least two Tau signatures that mimicked a CSA pattern found in a subset of aged TgP301L mice. Toxicity above baseline in transfected cells was greatest with brain-derived material, with different preparations differing in their kinetics of cell loss and fluorescent signals at the nuclear margin.

**Conclusions:** Heterogeneities in tau pathology and chemical signatures were observed within congenic stocks of TgP301L mice and in different P301L patients; crucially, signatures present in patients also manifested in
<figure><img src="image_84.png" /></figure>

a subset of mice. We conclude that our Tg model reveals non-synchronous, stochastic chemical events that initiate tau deposition and that different toxicity profiles of Tau isolates affect their ability to propagate.

**128. Strain profiles of CJD in venison consumers and non-consumers from Alberta and Saskatchewan**

Jennifer Myskiw<sup>a,b</sup>, Lise Lamourieux<sup>a</sup>, Michael Coulthart<sup>c</sup>, Valerie Sim<sup>d</sup> and Stephanie Booth<sup>a,b</sup>

<sup>a</sup>Zoonotic Diseases and Special Pathogens, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg; <sup>b</sup>Department of Medical Microbiology and Infectious Diseases, University of Manitoba, Winnipeg; <sup>c</sup>Canadian CJD Surveillance System, Public Health Agency of Canada, Ottawa; <sup>d</sup>Division of Neurology, Department of Medicine Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton

CONTACT Jennifer Myskiw Myskiwj@myumanitoba.ca

**ABSTRACT**

Chronic wasting disease (CWD) is a fatal neurodegenerative disease that is spreading rapidly through wild cervid populations in Alberta and Saskatchewan. The epidemic of CWD in Canada not only has implications for tourism and hunting but it is also a concern over possible zoonotic transmission to humans who eat venison from infected deer. When bovine spongiform encephalopathy (BSE) reached epidemic proportions, variant CJD was identified as an acquired form of BSE due to the unique biochemical fingerprint of the pathologic prion protein (PrP). While the Canadian CJD Surveillance System (CJDSS) is staying vigilant to identify CWD human cases, it is impossible to know what the phenotype of human CWD prions would present with or whether they would be distinct from sporadic CJD cases. Additionally, there is currently no convenient laboratory model to study these differences experimentally. For these reasons we are undertaking a systematic analysis of the molecular diversity of CJD cases in patients who resided in Alberta and Saskatchewan at their time of death. We are comparing CJD profiles of venison consumers and non-consumers using a variety of clinical imaging, pathological and biochemical markers. Firstly, MRIs have been reviewed and neuropathology of over 40 patients for whom CJD was confirmed as the cause of death analysed. In this way we are able to select clinically affected areas for further biochemical analysis of prion protein. We will present data including our analysis to date of levels of protease sensitive and resistant prion protein, temperature and detergent denaturation profiles, and glycoform typing. By combining these methodologies, we intend

to extend the baseline CJD typing that is already completed by the CJDSS. Importantly we aim to increase our understanding of the human prion strains that affect the Canadian population. By doing so, we will establish a baseline for the identification of any future atypical CJD cases. For example, those that could arise as a result of exposure to CWD.

**129. Deregulation of SINE RNA levels in Alzheimer's disease patients**

Yubo Cheng<sup>a</sup>, Luke Saville<sup>a</sup>, Babita Gollen<sup>a</sup>, Mitchell Liam<sup>b</sup>, Christopher Isaac<sup>a</sup>, Jogender Mehla<sup>b</sup>, Mohajerani Majid<sup>b</sup> and Athanasios Zovoilis<sup>a,b</sup>

<sup>a</sup>Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Canada; <sup>b</sup>Department of Neuroscience, University of Lethbridge, Lethbridge, Canada

CONTACT Athanasios Zovoilis athanasios.zovoilis@uleth.ca

**ABSTRACT**

As the human life span increases, the number of people in Canada suffering from Alzheimer's disease (AD) is expected to rise dramatically. A number of studies have revealed that gene expression networks in AD are deregulated. However, the molecular mechanisms associated with this deregulation remain largely unknown. In a previous work, we have shown that mechanisms involving non protein-coding RNAs, such as Short Interspersed Nuclear Element (SINE) RNAs (Zovoilis et al., Cell 2016) are key in maintaining transcriptional homeostasis in cells. Recently, we showed that in mouse, RNA from one of the most frequent SINE repeat classes (B2 RNA) controls expression of immediate early genes (IEGs) through binding and inhibition of RNA Polymerase II. However, it remained unclear whether deregulation of this mechanism could underlie a pathological condition in human. Here, we applied an integrative RNA genomics and bioinformatics approach to dissect any connection between SINE RNAs and Alzheimer's disease. Our study revealed that SINE RNAs are subject to abnormal processing in human AD patients and are connected with deregulation of gene expression.

**130. Evaluation of the sensitivity of different RT-QuIC substrates in detecting and characterizing CWD prions in brains of Norwegian cervids**

Edoardo Bistaffa<sup>a</sup>, Tram Thu Vuong<sup>b</sup>, Linh Tran<sup>b</sup>, Federico Angelo Cazzaniga<sup>a</sup>, Giulia Salzano<sup>c</sup>, Giuseppe Legname<sup>c</sup>, Giorgio Giaccone<sup>a</sup>, Sylvie Benestad<sup>b</sup> and Fabio Moda<sup>a</sup>
<figure><img src="image_84.png" /></figure>

a subset of mice. We conclude that our Tg model reveals non-synchronous, stochastic chemical events that initiate tau deposition and that different toxicity profiles of Tau isolates affect their ability to propagate.

**128. Strain profiles of CJD in venison consumers and non-consumers from Alberta and Saskatchewan**

Jennifer Myskiw<sup>a,b</sup>, Lise Lamourieux<sup>a</sup>, Michael Coulthart<sup>c</sup>, Valerie Sim<sup>d</sup> and Stephanie Booth<sup>a,b</sup>

<sup>a</sup>Zoonotic Diseases and Special Pathogens, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg; <sup>b</sup>Department of Medical Microbiology and Infectious Diseases, University of Manitoba, Winnipeg; <sup>c</sup>Canadian CJD Surveillance System, Public Health Agency of Canada, Ottawa; <sup>d</sup>Division of Neurology, Department of Medicine Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton

CONTACT Jennifer Myskiw Myskiwj@myumanitoba.ca

**ABSTRACT**

Chronic wasting disease (CWD) is a fatal neurodegenerative disease that is spreading rapidly through wild cervid populations in Alberta and Saskatchewan. The epidemic of CWD in Canada not only has implications for tourism and hunting but it is also a concern over possible zoonotic transmission to humans who eat venison from infected deer. When bovine spongiform encephalopathy (BSE) reached epidemic proportions, variant CJD was identified as an acquired form of BSE due to the unique biochemical fingerprint of the pathologic prion protein (PrP). While the Canadian CJD Surveillance System (CJDSS) is staying vigilant to identify CWD human cases, it is impossible to know what the phenotype of human CWD prions would present with or whether they would be distinct from sporadic CJD cases. Additionally, there is currently no convenient laboratory model to study these differences experimentally. For these reasons we are undertaking a systematic analysis of the molecular diversity of CJD cases in patients who resided in Alberta and Saskatchewan at their time of death. We are comparing CJD profiles of venison consumers and non-consumers using a variety of clinical imaging, pathological and biochemical markers. Firstly, MRIs have been reviewed and neuropathology of over 40 patients for whom CJD was confirmed as the cause of death analysed. In this way we are able to select clinically affected areas for further biochemical analysis of prion protein. We will present data including our analysis to date of levels of protease sensitive and resistant prion protein, temperature and detergent denaturation profiles, and glycoform typing. By combining these methodologies, we intend

to extend the baseline CJD typing that is already completed by the CJDSS. Importantly we aim to increase our understanding of the human prion strains that affect the Canadian population. By doing so, we will establish a baseline for the identification of any future atypical CJD cases. For example, those that could arise as a result of exposure to CWD.

**129. Deregulation of SINE RNA levels in Alzheimer's disease patients**

Yubo Cheng<sup>a</sup>, Luke Saville<sup>a</sup>, Babita Gollen<sup>a</sup>, Mitchell Liam<sup>b</sup>, Christopher Isaac<sup>a</sup>, Jogender Mehla<sup>b</sup>, Mohajerani Majid<sup>b</sup> and Athanasios Zovoilis<sup>a,b</sup>

<sup>a</sup>Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Canada; <sup>b</sup>Department of Neuroscience, University of Lethbridge, Lethbridge, Canada

CONTACT Athanasios Zovoilis athanasios.zovoilis@uleth.ca

**ABSTRACT**

As the human life span increases, the number of people in Canada suffering from Alzheimer's disease (AD) is expected to rise dramatically. A number of studies have revealed that gene expression networks in AD are deregulated. However, the molecular mechanisms associated with this deregulation remain largely unknown. In a previous work, we have shown that mechanisms involving non protein-coding RNAs, such as Short Interspersed Nuclear Element (SINE) RNAs (Zovoilis et al., Cell 2016) are key in maintaining transcriptional homeostasis in cells. Recently, we showed that in mouse, RNA from one of the most frequent SINE repeat classes (B2 RNA) controls expression of immediate early genes (IEGs) through binding and inhibition of RNA Polymerase II. However, it remained unclear whether deregulation of this mechanism could underlie a pathological condition in human. Here, we applied an integrative RNA genomics and bioinformatics approach to dissect any connection between SINE RNAs and Alzheimer's disease. Our study revealed that SINE RNAs are subject to abnormal processing in human AD patients and are connected with deregulation of gene expression.

**130. Evaluation of the sensitivity of different RT-QuIC substrates in detecting and characterizing CWD prions in brains of Norwegian cervids**

Edoardo Bistaffa<sup>a</sup>, Tram Thu Vuong<sup>b</sup>, Linh Tran<sup>b</sup>, Federico Angelo Cazzaniga<sup>a</sup>, Giulia Salzano<sup>c</sup>, Giuseppe Legname<sup>c</sup>, Giorgio Giaccone<sup>a</sup>, Sylvie Benestad<sup>b</sup> and Fabio Moda<sup>a</sup>
<figure><img src="image_85.png" /></figure>

<sup>a</sup>Division of Neurology 5 and Neuropathology, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milano, Italy; <sup>b</sup>Norwegian Veterinary Institute, Oslo, Norway; <sup>c</sup>Laboratory of Prion Biology, Department of Neuroscience, Scuola Internazionale Superiore di Studi Avanzati (SISSA), Trieste, Italy

CONTACT Tram Thu Vuong tram.thu.vuong@vetinst.no

## ABSTRACT

Chronic wasting disease (CWD) is a highly contagious prion pathology affecting captive and free-ranging cervid populations. From its first description 50 years ago, CWD has been detected in United States, Canada, South Korea and, most recently, in Norway. At present, we do not know if these diseases are caused by the same prion strain or whether they spontaneously arose in different countries. CWD spreading among cervids is one of the most important issue for public health due to the ability of this agent to infect a large number of animals and the possible transmission to other animal species, including humans and ovine. Indeed, possible transmission of CWD to ovine that share, in some countries, the same geographic areas of CWD infected animals cannot be excluded. For this reason, it is of fundamental importance to develop a strategy allowing identification of CWD infected samples and verify their ability to propagate within the same or different species. To this aim, we are optimizing RT-QuIC assay coupled with specific biochemical analysis for evaluating whether different CWD samples could produce final reactions products with peculiar differences useful for CWD strain's identification. RT-QuIC were performed using different substrates of reaction (truncated PrP protein from hamster, reindeer and deer species) for evaluating their sensitivity and specificity in detecting CWD in brains of Norwegian moose, red deer and reindeer. Moreover, through biochemical assessments (PK digestion, guanidine hydrochloride evaluation and use of antibodies against different PrP epitopes), we evaluated whether and to what extent final RT-QuIC products were able to acquire distinct biochemical features according to the strain of CWD. Preliminary results showed that deer PrP detected CWD in all species with higher efficiency compared to hamster and reindeer. Notably, truncated deer-PrP was able to rapidly detect positive samples (within 3 h from the beginning of the reaction) compared to the other substrates. Although with less sensitivity, reindeer-PrP identified red deer, reindeer and moose CWD, with this latter being characterized by lower resistance to PK digestion and different PrP banding profile (at Western blot) compared to the others. Therefore, RT-QuIC can be exploited for detecting CWD infected species with high sensitivity and specificity, while biochemical data seem to indicate the possibility of identifying the species of CWD origin. If confirmed, such information will be of fundamental importance for planning

actions to contain the spread of the infection (especially to other species, like ovines) in CWD affected regions.

**KEYWORDS:** Prion; CWD; RT-QuIC

# 131. Targeting a regulatory subunit of protein phosphatase 1 by Sephin-1 reduces prion infection in neuronal cells

Simrika Thapa, Dalia. H. Abdelaziz, Basant Abdulrahman and Hermann M. Schatzl

Department of Comparative Biology & Experimental Medicine, Hotchkiss Brain Institute and Calgary Prion Research Unit, University of Calgary, Calgary, Alberta, Canada

CONTACT Simrika Thapa sthapa@ucalgary.ca

## ABSTRACT

Prion diseases are infectious and fatal neurodegenerative diseases in human and animals caused by misfolding of the cellular prion protein (PrP<sup>C</sup>) into the infectious isoform PrP<sup>Sc</sup>. These diseases have the potential to transmit within or between species, and no cure is available to date. Targeting the unfolded protein response (UPR) as an anti-prion therapeutic approach has been widely reported for prion diseases. Here, we describe the anti-prion effect of Sephin-1 which has been shown to protect in mouse models of protein misfolding diseases including amyotrophic lateral sclerosis by selectively inhibiting the stress-induced regulatory subunit of protein phosphatase 1, thus prolonging the eIF2α phosphorylation. In this study, we describe that Sephin-1 significantly reduced PrP<sup>Sc</sup> in persistently prion-infected neuronal cells, 22L-ScN2a, RML-ScN2a and RML-ScCAD5 cells. Moreover, prion seeding activity was reduced in treated cells in RT-QuIC assay. No effect of Sephin-1 on the levels of PrP<sup>C</sup> expression was observed in uninfected N2a cells. Importantly, we found that Sephin-1 significantly overcame the ER stress induced in treated cells as measured by lower expression levels of stress-induced aberrant PrP and stress markers (BiP and CHOP). At present, we are investigating effectiveness of intraperitoneal Sephin-1 treatment against RML prions in vivo. These results suggest that Sephin-1 could be a potential anti-prion drug selectively targeting one component of UPR pathway.

# 132. Metformin ameliorates prion infection in neuronal cells by enhancing autophagy

Dalia Abdelaziz, Simrika Thapa, Basant Abdulrahman and Hermann Schatzl
<figure><img src="image_85.png" /></figure>

<sup>a</sup>Division of Neurology 5 and Neuropathology, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milano, Italy; <sup>b</sup>Norwegian Veterinary Institute, Oslo, Norway; <sup>c</sup>Laboratory of Prion Biology, Department of Neuroscience, Scuola Internazionale Superiore di Studi Avanzati (SISSA), Trieste, Italy

CONTACT Tram Thu Vuong tram.thu.vuong@vetinst.no

## ABSTRACT

Chronic wasting disease (CWD) is a highly contagious prion pathology affecting captive and free-ranging cervid populations. From its first description 50 years ago, CWD has been detected in United States, Canada, South Korea and, most recently, in Norway. At present, we do not know if these diseases are caused by the same prion strain or whether they spontaneously arose in different countries. CWD spreading among cervids is one of the most important issue for public health due to the ability of this agent to infect a large number of animals and the possible transmission to other animal species, including humans and ovine. Indeed, possible transmission of CWD to ovine that share, in some countries, the same geographic areas of CWD infected animals cannot be excluded. For this reason, it is of fundamental importance to develop a strategy allowing identification of CWD infected samples and verify their ability to propagate within the same or different species. To this aim, we are optimizing RT-QuIC assay coupled with specific biochemical analysis for evaluating whether different CWD samples could produce final reactions products with peculiar differences useful for CWD strain's identification. RT-QuIC were performed using different substrates of reaction (truncated PrP protein from hamster, reindeer and deer species) for evaluating their sensitivity and specificity in detecting CWD in brains of Norwegian moose, red deer and reindeer. Moreover, through biochemical assessments (PK digestion, guanidine hydrochloride evaluation and use of antibodies against different PrP epitopes), we evaluated whether and to what extent final RT-QuIC products were able to acquire distinct biochemical features according to the strain of CWD. Preliminary results showed that deer PrP detected CWD in all species with higher efficiency compared to hamster and reindeer. Notably, truncated deer-PrP was able to rapidly detect positive samples (within 3 h from the beginning of the reaction) compared to the other substrates. Although with less sensitivity, reindeer-PrP identified red deer, reindeer and moose CWD, with this latter being characterized by lower resistance to PK digestion and different PrP banding profile (at Western blot) compared to the others. Therefore, RT-QuIC can be exploited for detecting CWD infected species with high sensitivity and specificity, while biochemical data seem to indicate the possibility of identifying the species of CWD origin. If confirmed, such information will be of fundamental importance for planning

actions to contain the spread of the infection (especially to other species, like ovines) in CWD affected regions.

**KEYWORDS:** Prion; CWD; RT-QuIC

## 131. Targeting a regulatory subunit of protein phosphatase 1 by Sephin-1 reduces prion infection in neuronal cells

Simrika Thapa, Dalia. H. Abdelaziz, Basant Abdulrahman and Hermann M. Schatzl

Department of Comparative Biology & Experimental Medicine, Hotchkiss Brain Institute and Calgary Prion Research Unit, University of Calgary, Calgary, Alberta, Canada

CONTACT Simrika Thapa sthapa@ucalgary.ca

## ABSTRACT

Prion diseases are infectious and fatal neurodegenerative diseases in human and animals caused by misfolding of the cellular prion protein (PrP<sup>C</sup>) into the infectious isoform PrP<sup>Sc</sup>. These diseases have the potential to transmit within or between species, and no cure is available to date. Targeting the unfolded protein response (UPR) as an anti-prion therapeutic approach has been widely reported for prion diseases. Here, we describe the anti-prion effect of Sephin-1 which has been shown to protect in mouse models of protein misfolding diseases including amyotrophic lateral sclerosis by selectively inhibiting the stress-induced regulatory subunit of protein phosphatase 1, thus prolonging the eIF2α phosphorylation. In this study, we describe that Sephin-1 significantly reduced PrP<sup>Sc</sup> in persistently prion-infected neuronal cells, 22L-ScN2a, RML-ScN2a and RML-ScCAD5 cells. Moreover, prion seeding activity was reduced in treated cells in RT-QuIC assay. No effect of Sephin-1 on the levels of PrP<sup>C</sup> expression was observed in uninfected N2a cells. Importantly, we found that Sephin-1 significantly overcame the ER stress induced in treated cells as measured by lower expression levels of stress-induced aberrant PrP and stress markers (BiP and CHOP). At present, we are investigating effectiveness of intraperitoneal Sephin-1 treatment against RML prions in vivo. These results suggest that Sephin-1 could be a potential anti-prion drug selectively targeting one component of UPR pathway.

## 132. Metformin ameliorates prion infection in neuronal cells by enhancing autophagy

Dalia Abdelaziz, Simrika Thapa, Basant Abdulrahman and Hermann Schatzl
<figure><img src="image_86.png" /></figure>

Department Comparative Biology & Experimental Medicine, Faculty of Veterinary Medicine, Calgary Prion Research Unit, and Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta, Canada.

CONTACT Dalia Abdelaziz dalia.abdelaziz@ucalgary.ca

## ABSTRACT

Metformin is the first-line medication for the treatment of type 2 diabetes and a well-known AMPK activator. Recently, substantial efforts have been invested in establishing the role of metformin in the treatment of neurodegenerative diseases, such as Alzheimer's, amnestic mild cognitive impairment, Parkinson's and Huntington's disease. Metformin was shown to be neuroprotective and to promote adult neurogenesis under both physiological and pathological conditions in vivo. This prompted us to investigate the anti-prion effectiveness of metformin in two different persistently prion infected neuronal cell lines (RML-CAD5 and 22L-N2a). We found that metformin treatment, at non-toxic concentrations of 0.5 and 1 mM, decreased the $PrP^{Sc}$ load in both 22L-N2a and RML-CAD5 cells, as shown by lower signals of PK resistant PrP in immunoblots and less conversion activity in RT-QuIC. On the other hand, metformin treatment had no effect on $PrP^C$ levels in uninfected N2a cells. Since there is a solid body of evidence showing autophagy enhancing activity of metformin, we decided to test the level of autophagy markers in the treated cells. Notably, the LC3 II signal was found to be upregulated in both CAD5 and N2a cells after treatment with metformin, which confirms the autophagy inducing effect of metformin in both neuronal cell lines. Currently, we are investigating the effect of intraperitoneal metformin treatment on the survival of RML-infected mice. In conclusion, our findings describe for the first time the anti-prion effect of metformin, which can be at least partially explained by its autophagy inducing activity. In future studies, we are planning to do synergistic combination of metformin with other anti-prion candidate drugs to test if this will improve the anti-prion efficacy via working on different pathways.

## 133. Neurofilaments in blood: a new promising biomarker of scrapie

Marco Rossiª, Elena Bozzettaª, Cristina Casaloneª, Cristiano Coronaª, Maria C. Cavarrettaª, Henrik Zetterbergᵇ, Kaj Blennowᵇ and Daniela Meloniª

CONTACT Marco Rossi marco.rossi@izsto.it

## ABSTRACT

**Introduction:** *In vivo* accurate, easy-to-perform, and early diagnostic tests are crucial for the identification of prion subjects during their early or preclinical phase of disease and finally for managing public health issues. Excellent biomarkers for prion diseases have been identified in the cerebrospinal fluid (CSF), but this tissue is difficult to get from sheep and is therefore of little utility for the control of scrapie. Recently, the measurement of neurofilaments light chain (NF-L), a surrogate biomarker of neuroaxonal degeneration, by the SIMOA ultra-sensitive single molecule array technology, revealed high NF-L levels in blood of patients with preclinical stages of human prion diseases. In this work, we adapted the SIMOA test for the measurement of NF-L in blood of scrapie infected sheep and healthy animals and it showed a promising utility of blood serum NF-L as biomarker of scrapie in sheep.

**Materials and Methods:** A set of 20 sheep blood serum blinded samples were tested by the SIMOA technology. Nine samples were from scrapie affected sheep and 11 from healthy animals. The gold standard was assessed by testing the *medulla oblongata* of sheep both with an EU approved Elisa rapid test followed by a confirmatory Western blot assay.

**Results:** Results revealed a clear capacity of Simoa NFL test to classify the two groups of samples according to the presence or absence of scrapie infection. All the positive samples according to the gold standard showed high NF-L levels, while only one of 10 samples from healthy animals showed NF-L values overlapping with positive samples.

**Conclusion:** These data suggest that neurofilaments are sensitive and specific blood biomarkers for the diagnosis of scrapie in sheep and might represent promising tools for an early diagnosis. We are now working for establishing the basal levels of NF-L in a larger group of healthy sheep and in sheep with different types of diseases other than scrapie. We are also working for determining the variation of NF-L levels in experimentally scrapie-infected sheep at various intervals of the incubation period and at the terminal stages of disease.

## 134. The transmissible spongiform encephalopathies surveillance in small ruminants in Romania for a period of 10 years

Florica Bărbuceanuª, Ioana Neghirlaᵇ, Theodora Chesnoiuᵇ, Cristina Diaconuª, Stefania-Felicia Barbuceanuᶜ and G. Predoiᵈ

<footnote>ªIstituto zooprofilattico del Piemonte Liguria e Valle d'Aosta, Turin, Italy; ᵇInstitute of neuroscience and physiology, University of Gothenburg, Gothenburg, Sweden</footnote>
<figure><img src="image_86.png" /></figure>

Department Comparative Biology & Experimental Medicine, Faculty of Veterinary Medicine, Calgary Prion Research Unit, and Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta, Canada.

CONTACT Dalia Abdelaziz dalia.abdelaziz@ucalgary.ca

## ABSTRACT

Metformin is the first-line medication for the treatment of type 2 diabetes and a well-known AMPK activator. Recently, substantial efforts have been invested in establishing the role of metformin in the treatment of neurodegenerative diseases, such as Alzheimer's, amnestic mild cognitive impairment, Parkinson's and Huntington's disease. Metformin was shown to be neuroprotective and to promote adult neurogenesis under both physiological and pathological conditions in vivo. This prompted us to investigate the anti-prion effectiveness of metformin in two different persistently prion infected neuronal cell lines (RML-CAD5 and 22L-N2a). We found that metformin treatment, at non-toxic concentrations of 0.5 and 1 mM, decreased the $PrP^{Sc}$ load in both 22L-N2a and RML-CAD5 cells, as shown by lower signals of PK resistant PrP in immunoblots and less conversion activity in RT-QuIC. On the other hand, metformin treatment had no effect on $PrP^C$ levels in uninfected N2a cells. Since there is a solid body of evidence showing autophagy enhancing activity of metformin, we decided to test the level of autophagy markers in the treated cells. Notably, the LC3 II signal was found to be upregulated in both CAD5 and N2a cells after treatment with metformin, which confirms the autophagy inducing effect of metformin in both neuronal cell lines. Currently, we are investigating the effect of intraperitoneal metformin treatment on the survival of RML-infected mice. In conclusion, our findings describe for the first time the anti-prion effect of metformin, which can be at least partially explained by its autophagy inducing activity. In future studies, we are planning to do synergistic combination of metformin with other anti-prion candidate drugs to test if this will improve the anti-prion efficacy via working on different pathways.

## 133. Neurofilaments in blood: a new promising biomarker of scrapie

Marco Rossiª, Elena Bozzettaª, Cristina Casaloneª, Cristiano Coronaª, Maria C. Cavarrettaª, Henrik Zetterbergᵇ, Kaj Blennowᵇ and Daniela Meloniª

CONTACT Marco Rossi marco.rossi@izsto.it

## ABSTRACT

**Introduction:** *In vivo* accurate, easy-to-perform, and early diagnostic tests are crucial for the identification of prion subjects during their early or preclinical phase of disease and finally for managing public health issues. Excellent biomarkers for prion diseases have been identified in the cerebrospinal fluid (CSF), but this tissue is difficult to get from sheep and is therefore of little utility for the control of scrapie. Recently, the measurement of neurofilaments light chain (NF-L), a surrogate biomarker of neuroaxonal degeneration, by the SIMOA ultra-sensitive single molecule array technology, revealed high NF-L levels in blood of patients with preclinical stages of human prion diseases. In this work, we adapted the SIMOA test for the measurement of NF-L in blood of scrapie infected sheep and healthy animals and it showed a promising utility of blood serum NF-L as biomarker of scrapie in sheep.

**Materials and Methods:** A set of 20 sheep blood serum blinded samples were tested by the SIMOA technology. Nine samples were from scrapie affected sheep and 11 from healthy animals. The gold standard was assessed by testing the *medulla oblongata* of sheep both with an EU approved Elisa rapid test followed by a confirmatory Western blot assay.

**Results:** Results revealed a clear capacity of Simoa NFL test to classify the two groups of samples according to the presence or absence of scrapie infection. All the positive samples according to the gold standard showed high NF-L levels, while only one of 10 samples from healthy animals showed NF-L values overlapping with positive samples.

**Conclusion:** These data suggest that neurofilaments are sensitive and specific blood biomarkers for the diagnosis of scrapie in sheep and might represent promising tools for an early diagnosis. We are now working for establishing the basal levels of NF-L in a larger group of healthy sheep and in sheep with different types of diseases other than scrapie. We are also working for determining the variation of NF-L levels in experimentally scrapie-infected sheep at various intervals of the incubation period and at the terminal stages of disease.

## 134. The transmissible spongiform encephalopathies surveillance in small ruminants in Romania for a period of 10 years

Florica Bărbuceanuª, Ioana Neghirlaᵇ, Theodora Chesnoiuᵇ, Cristina Diaconuª, Stefania-Felicia Barbuceanuᶜ and G. Predoiᵈ

<footnote>ªIstituto zooprofilattico del Piemonte Liguria e Valle d'Aosta, Turin, Italy; ᵇInstitute of neuroscience and physiology, University of Gothenburg, Gothenburg, Sweden</footnote>
<figure><img src="image_87.png" /></figure>

<sup>a</sup>Institute for Diagnosis and Animal Health Bucharest, Bucureşti, Romania; <sup>b</sup>National Sanitary Veterinary and Food Safety Authority, Bucureşti, Romania; <sup>c</sup>Faculty of Pharmacy, Carol Davila University of Medicine and Pharmacy, Bucureşti, Romania; <sup>d</sup>Faculty of Veterinary Medicine Bucharest, Bucureşti, Romania

CONTACT Florica Bărbuceanu barbuceanu.florica@idah.ro

## ABSTRACT

The scarpie belongs to the TSE group and is a fatal degenerative disease, affecting the central nervous system of ovine and caprine. There are two types of scrapie: classical and atypical. The classical scrapie affects the animals aged from 2 to 5 years and is very contagious, while the atypical scrapie affects the animals older than 5 years and is considered to have a low degree of infectivity. The aim of this study is to present information on the Romanian TSE Surveillance Program, performed in compliance with the EU Commission and OIE requirements, as well as the TSE test results in small ruminants (animals of the category clinical suspicions, dead animals and animals slaughtered normally) performed in the national net for transmissible spongiform encephalopathies, between 2007 and 2017. Between 2007 and 2017 were tested in Romania approximatively 339,643 small ruminants. All the 787 cases of scrapie were confirmed by the TSE NRL (National Reference Laboratory) by immunoblotting and immunohistochemical tests, with the performance of the discriminatory and genotyping testing. All animals diagnosed with scrapie were affected by the classical form. For all suspicion cases, a differential diagnosis was performed against rabies, Aujeszky disease and listeriosis.

**KEYWORDS:** Classical scrapie; Romanian TSE surveillance program; NRL-TSE

## 135. Characterization of serum miRNA in prion infected elk and hamsters reveal putative biomarkers

Jessy Slota<sup>a</sup>, Sarah Medina<sup>b</sup>, Megan Klassen<sup>c</sup> and
Stephanie Booth<sup>b</sup>

<sup>a</sup>University of Manitoba, Winnipeg, Canada; <sup>b</sup>Public Health Agency of Canada, Ottawa, Canada; <sup>c</sup>Public Health Agency of Canada - NML

CONTACT Jessy Slota slotaj@myumanitoba.ca

## ABSTRACT

Prion diseases are a group of fatal neurodegenerative diseases that arise in both humans and animals following misfolding of the cellular prion protein into a disease

associated structural conformation. Afflicted individuals experience a decline in mental capacity owing to the death of brain cells and spongiform change, although the phenotypic characteristics of these diseases vary widely. This heterogeneity as well as a lack of robust diagnostic methods make the diagnosis of prion diseases very challenging. It is especially important to develop better diagnostic assays for prion diseases as diagnosis must precede the onset of clinical signs and symptoms as well as permanent neuronal damage for any potential therapeutics to be of benefit. Micro-RNAs (miRNAs) are small non-coding RNA molecules which regulate expression of mRNA through the RNA interference pathway and may be found within cells as well as circulating biological fluids such as serum and cerebrospinal fluid (CSF). Identification of signatures of altered levels of specific miRNAs within biofluids during disease states is a promising new avenue of research which may fuel the development of improved diagnostic assays. Therefore, identifying patterns of miRNAs deregulated within biofluids such as serum during prion infection may lead to the discovery of novel biomarkers, aiding in the diagnosis of these diseases. In the present study, Illumina next-generation sequencing was used to characterize the miRNA content of serum taken from Elk with chronic wasting disease (CWD) as well as hamsters infected with 263K scrapie prions. This analysis was also applied to CSF taken from the 263K infected hamsters. Multiple miRNAs were detected to be altered between both serum and CSF isolated from the infected animals compared to the healthy control animals. Subsequent analysis revealed a pattern of five miRNAs which were consistently down-regulated in serum samples taken from both elk and hamsters infected with prions, and therefore are strong candidate biomarkers for prion diseases. These results may lay the groundwork towards utilizing altered miRNA signatures as the basis for improved prion diagnostic assays.

## 136. Identification of novel microRNA candidates mediating alpha-synuclein uptake in cells

Elena De Cecco<sup>a</sup>, Remo Sanges<sup>b</sup>, Luca Braga<sup>c</sup>, Paola Zago<sup>a</sup>, Joanna Narkiewicz<sup>a</sup>, Fabio Perissinotto<sup>c</sup>, Irene Guadagnino<sup>e</sup>, Sandro Banfi<sup>e</sup>, Mauro Giacca<sup>c</sup> and Giuseppe Legname<sup>a,d</sup>

<sup>a</sup>Laboratory of Prion Biology, Department of Neuroscience, Scuola Internazionale Superiore di Studi Avanzati (SISSA), Trieste, Italy; <sup>b</sup>Computational Genomics Laboratory, Department of Neuroscience, Scuola Internazionale Superiore di Studi Avanzati (SISSA), Trieste, Italy; <sup>c</sup>ICGEB International Centre for Genetic Engineering and Biotechnology, Padriciano, Trieste, Italy; <sup>d</sup>ELETTRA
<figure><img src="image_87.png" /></figure>

<sup>a</sup>Institute for Diagnosis and Animal Health Bucharest, Bucureşti, Romania; <sup>b</sup>National Sanitary Veterinary and Food Safety Authority, Bucureşti, Romania; <sup>c</sup>Faculty of Pharmacy, Carol Davila University of Medicine and Pharmacy, Bucureşti, Romania; <sup>d</sup>Faculty of Veterinary Medicine Bucharest, Bucureşti, Romania

CONTACT Florica Bărbuceanu barbuceanu.florica@idah.ro

## ABSTRACT

The scarpie belongs to the TSE group and is a fatal degenerative disease, affecting the central nervous system of ovine and caprine. There are two types of scrapie: classical and atypical. The classical scrapie affects the animals aged from 2 to 5 years and is very contagious, while the atypical scrapie affects the animals older than 5 years and is considered to have a low degree of infectivity. The aim of this study is to present information on the Romanian TSE Surveillance Program, performed in compliance with the EU Commission and OIE requirements, as well as the TSE test results in small ruminants (animals of the category clinical suspicions, dead animals and animals slaughtered normally) performed in the national net for transmissible spongiform encephalopathies, between 2007 and 2017. Between 2007 and 2017 were tested in Romania approximatively 339,643 small ruminants. All the 787 cases of scrapie were confirmed by the TSE NRL (National Reference Laboratory) by immunoblotting and immunohistochemical tests, with the performance of the discriminatory and genotyping testing. All animals diagnosed with scrapie were affected by the classical form. For all suspicion cases, a differential diagnosis was performed against rabies, Aujeszky disease and listeriosis.

**KEYWORDS:** Classical scrapie; Romanian TSE surveillance program; NRL-TSE

## **135. Characterization of serum miRNA in prion infected elk and hamsters reveal putative biomarkers**

Jessy Slota<sup>a</sup>, Sarah Medina<sup>b</sup>, Megan Klassen<sup>c</sup> and
Stephanie Booth<sup>b</sup>

<sup>a</sup>University of Manitoba, Winnipeg, Canada; <sup>b</sup>Public Health Agency of Canada, Ottawa, Canada; <sup>c</sup>Public Health Agency of Canada - NML

CONTACT Jessy Slota slotaj@myumanitoba.ca

## ABSTRACT

Prion diseases are a group of fatal neurodegenerative diseases that arise in both humans and animals following misfolding of the cellular prion protein into a disease

associated structural conformation. Afflicted individuals experience a decline in mental capacity owing to the death of brain cells and spongiform change, although the phenotypic characteristics of these diseases vary widely. This heterogeneity as well as a lack of robust diagnostic methods make the diagnosis of prion diseases very challenging. It is especially important to develop better diagnostic assays for prion diseases as diagnosis must precede the onset of clinical signs and symptoms as well as permanent neuronal damage for any potential therapeutics to be of benefit. Micro-RNAs (miRNAs) are small non-coding RNA molecules which regulate expression of mRNA through the RNA interference pathway and may be found within cells as well as circulating biological fluids such as serum and cerebrospinal fluid (CSF). Identification of signatures of altered levels of specific miRNAs within biofluids during disease states is a promising new avenue of research which may fuel the development of improved diagnostic assays. Therefore, identifying patterns of miRNAs deregulated within biofluids such as serum during prion infection may lead to the discovery of novel biomarkers, aiding in the diagnosis of these diseases. In the present study, Illumina next-generation sequencing was used to characterize the miRNA content of serum taken from Elk with chronic wasting disease (CWD) as well as hamsters infected with 263K scrapie prions. This analysis was also applied to CSF taken from the 263K infected hamsters. Multiple miRNAs were detected to be altered between both serum and CSF isolated from the infected animals compared to the healthy control animals. Subsequent analysis revealed a pattern of five miRNAs which were consistently down-regulated in serum samples taken from both elk and hamsters infected with prions, and therefore are strong candidate biomarkers for prion diseases. These results may lay the groundwork towards utilizing altered miRNA signatures as the basis for improved prion diagnostic assays.

## **136. Identification of novel microRNA candidates mediating alpha-synuclein uptake in cells**

Elena De Cecco<sup>a</sup>, Remo Sanges<sup>b</sup>, Luca Braga<sup>c</sup>, Paola Zago<sup>a</sup>, Joanna Narkiewicz<sup>a</sup>, Fabio Perissinotto<sup>c</sup>, Irene Guadagnino<sup>e</sup>, Sandro Banfi<sup>e</sup>, Mauro Giacca<sup>c</sup> and Giuseppe Legname<sup>a,d</sup>

<footnote><sup>a</sup>Laboratory of Prion Biology, Department of Neuroscience, Scuola Internazionale Superiore di Studi Avanzati (SISSA), Trieste, Italy;
<sup>b</sup>Computational Genomics Laboratory, Department of Neuroscience, Scuola Internazionale Superiore di Studi Avanzati (SISSA), Trieste, Italy; <sup>c</sup>ICGEB International Centre for Genetic Engineering and Biotechnology, Padriciano, Trieste, Italy; <sup>d</sup>ELETTRA</footnote>
<figure><img src="image_88.png" /></figure>

Sincrotrone Trieste S.C.p.A, Basovizza, Trieste, Italy; eTIGEM Telethon Institute of Genetics and Medicine, Pozzuoli, Napoli, Italy.

CONTACT Elena De Cecco edececco@sissa.it

## ABSTRACT

**Background:** Alpha-synucleinopathies are a group of fatal neurodegenerative disorders for which neither effective therapies nor preclinical biomarkers are yet available. Indeed, one major characteristic of these syndromes is that the first clinical symptoms start to appear only when more than 70% of dopaminergic neurons in the *substantia nigra* and striatum have been lost, and brain functions are irreversibly compromised. Hence, the identification of early-stage biomarkers poses as a crucial goal in the fight against the progression of neurodegeneration, as well as for the development of more effective and specific drugs. Dysregulation of the expression of non-coding RNAs (ncRNAs) is a common feature among several neurodegenerative diseases. Micro RNAs (miRNAs) are the most studied among ncRNAs and have been shown to participate in Multiple System Atrophy and Parkinson's Disease pathogenesis at various levels. This work aims at identifying miRNAs that play a role in the internalization of extracellular alpha-synuclein aggregates, which is a key step in the propagation of synucleinopathies.

**Materials and methods:** Our analysis focuses on the internalization of short fibrillar species of alpha-synuclein, as recent findings suggest that short fibres are easily internalized by cells and mediate the spreading of the pathology. To this aim, we set up a comprehensive miRNA screening approach that takes advantage of high throughput screening technology and allows us to screen thousands of miRNAs at the same time. The evaluation of the effect of each miRNA is based on the differential fluorescent labelling of internalized and non-internalized aggregates.

**Results:** Starting from a library of 2042 miRNAs, we found several candidates, which, upon overexpression, either promote or inhibit the internalization of alpha-synuclein short fibrils. Top hits from each phenotype were subjected to a detailed bioinformatics analysis resulting in the identification of multiple possible target genes, many of which are common targets of more top hit miRNAs. We are currently validating the role of some of the selected target genes in order to unravel the contribution of new molecular pathways to the complex phenomenon of aggregates propagation.

**Conclusions:** Our approach led to the identification of a number of miRNAs involved in alpha-synuclein short

fibrils uptake, as well as of some target genes. These findings represent a significant advancement to better understand the complex miRNA regulation in diseased brains, and will eventually provide a starting point to design miRNA-based therapies against synucleinopathies.

# 137. Stem cells treatment ameliorate clinical signs and increase incubation periods in prion infected rodents

Paulina Soto, Enrique Antonio Armijo Fuentes, Carlos Kramm, Claudio Soto and Rodrigo Morales

Department of Neurology, The University of Texas Health Science Center at Houston, Houston, TX, USA.

CONTACT Paulina Soto paulina.i.sotosoto@uth.tmc.edu

## ABSTRACT

Prion diseases or transmissible spongiform encephalopathies are a family of rare progressive neurodegenerative disorders that affect both humans and animals. Currently, these disorders have no cure. In the last decades, great efforts have been made to establish effective disease-modifying options to treat them. In recent years, the potential use of induce pluripotent stem cells (iPS) derived neuroprecursors (NPs) has been suggested as a promising cell-replacement therapy for neurodegenerative disorders. iPS-derived NPs have been shown to be effective in animal models of several neurodegenerative diseases including Parkinson's and Huntington's diseases, multiple sclerosis, and amyotrophic lateral sclerosis. We tested the therapeutic potential of NPs on prion infected rodents using different means of delivery. Briefly, C57BL/6J female mice were intra-cranially inoculated with RML prions and the potential benefits of NPs was evaluated in three different formats: (1) intra-cerebral administration when the animals showed the first prion-associated clinical signs, (2) intra-venous treatment before the onset of symptoms, and (3) i.v infusions on early symptomatic subjects. Our results show that in all cases NPs significantly extended the clinical phase and incubation periods of prions. Interestingly, peripheral administration of NPs-derived supernatants ameliorated the clinical signs associated to prion disease; however, did not have effect on the incubation periods. Our findings suggest that intracerebral and peripheral administration of iPS-derived NPs might be potentially used to treat prion diseases.
<figure><img src="image_88.png" /></figure>

Sincrotrone Trieste S.C.p.A, Basovizza, Trieste, Italy; eTIGEM Telethon Institute of Genetics and Medicine, Pozzuoli, Napoli, Italy.

CONTACT Elena De Cecco edececco@sissa.it

## ABSTRACT

**Background:** Alpha-synucleinopathies are a group of fatal neurodegenerative disorders for which neither effective therapies nor preclinical biomarkers are yet available. Indeed, one major characteristic of these syndromes is that the first clinical symptoms start to appear only when more than 70% of dopaminergic neurons in the *substantia nigra* and striatum have been lost, and brain functions are irreversibly compromised. Hence, the identification of early-stage biomarkers poses as a crucial goal in the fight against the progression of neurodegeneration, as well as for the development of more effective and specific drugs. Dysregulation of the expression of non-coding RNAs (ncRNAs) is a common feature among several neurodegenerative diseases. Micro RNAs (miRNAs) are the most studied among ncRNAs and have been shown to participate in Multiple System Atrophy and Parkinson's Disease pathogenesis at various levels. This work aims at identifying miRNAs that play a role in the internalization of extracellular alpha-synuclein aggregates, which is a key step in the propagation of synucleinopathies.

**Materials and methods:** Our analysis focuses on the internalization of short fibrillar species of alpha-synuclein, as recent findings suggest that short fibres are easily internalized by cells and mediate the spreading of the pathology. To this aim, we set up a comprehensive miRNA screening approach that takes advantage of high throughput screening technology and allows us to screen thousands of miRNAs at the same time. The evaluation of the effect of each miRNA is based on the differential fluorescent labelling of internalized and non-internalized aggregates.

**Results:** Starting from a library of 2042 miRNAs, we found several candidates, which, upon overexpression, either promote or inhibit the internalization of alpha-synuclein short fibrils. Top hits from each phenotype were subjected to a detailed bioinformatics analysis resulting in the identification of multiple possible target genes, many of which are common targets of more top hit miRNAs. We are currently validating the role of some of the selected target genes in order to unravel the contribution of new molecular pathways to the complex phenomenon of aggregates propagation.

**Conclusions:** Our approach led to the identification of a number of miRNAs involved in alpha-synuclein short

fibrils uptake, as well as of some target genes. These findings represent a significant advancement to better understand the complex miRNA regulation in diseased brains, and will eventually provide a starting point to design miRNA-based therapies against synucleinopathies.

# 137. Stem cells treatment ameliorate clinical signs and increase incubation periods in prion infected rodents

Paulina Soto, Enrique Antonio Armijo Fuentes, Carlos Kramm, Claudio Soto and Rodrigo Morales

Department of Neurology, The University of Texas Health Science Center at Houston, Houston, TX, USA.

CONTACT Paulina Soto paulina.i.sotosoto@uth.tmc.edu

## ABSTRACT

Prion diseases or transmissible spongiform encephalopathies are a family of rare progressive neurodegenerative disorders that affect both humans and animals. Currently, these disorders have no cure. In the last decades, great efforts have been made to establish effective disease-modifying options to treat them. In recent years, the potential use of induce pluripotent stem cells (iPS) derived neuroprecursors (NPs) has been suggested as a promising cell-replacement therapy for neurodegenerative disorders. iPS-derived NPs have been shown to be effective in animal models of several neurodegenerative diseases including Parkinson's and Huntington's diseases, multiple sclerosis, and amyotrophic lateral sclerosis. We tested the therapeutic potential of NPs on prion infected rodents using different means of delivery. Briefly, C57BL/6J female mice were intra-cranially inoculated with RML prions and the potential benefits of NPs was evaluated in three different formats: (1) intra-cerebral administration when the animals showed the first prion-associated clinical signs, (2) intra-venous treatment before the onset of symptoms, and (3) i.v infusions on early symptomatic subjects. Our results show that in all cases NPs significantly extended the clinical phase and incubation periods of prions. Interestingly, peripheral administration of NPs-derived supernatants ameliorated the clinical signs associated to prion disease; however, did not have effect on the incubation periods. Our findings suggest that intracerebral and peripheral administration of iPS-derived NPs might be potentially used to treat prion diseases.
<figure><img src="image_89.png" /></figure>

**138. Human cerebral organoids propagate sporadic CJD prions**

Bradley R. Grovemanª, Simote Foliakiª, Christina D. Orruª, Gianluigi Zanussoᵇ, James A. Carrollª, Brent Raceª and Kathryn L. Haighª

CONTACT Kathryn L. Haigh [cathryn.haigh@nih.gov](mailto:cathryn.haigh@nih.gov)

ABSTRACT

**Introduction:** Human prion disease has traditionally been difficult to model in cell culture. Very few human cell lines show sustained prion propagation over time and none represent three-dimensional (3D) brain tissue. Recently it was found that astrocyte cultures differentiated from human induced pluripotent stem cells (Hu-iPSCs) take up and propagate sporadic CJD (sCJD) prions with the resulting infection influenced by the sCJD subtype [1]. Hu-iPSCs have also been utilized to differentiate self-patterning, structured human brain tissues called cerebral organoids [2,3]. We hypothesized that these cerebral organoids could be used to model human prion propagation *in vitro*.

**Materials and Methods:** Cerebral organoid cultures were generated from hu-iPSCs using the protocol developed by Lancaster and Knoblich [2]. Cerebral organoids were infected with one of two sCJD inocula and monitored for health, morphology, PrP seeding activity, PrP deposition and proteinase-K resistant PrP as compared with organoids that received a normal brain homogenate inoculum.

**Results:** Cerebral organoids showed uptake and propagation of prions, detected by RT-QuIC analysis, following exposure to either sCJD inocula. Inoculum specific differences were observed. One inoculum showed robust proteinase-K resistant PrP in all tested organoids and coarse, granular PrP staining in the organoid interiors. The other inoculum induced a more toxic phenotype that affected organoid health but displayed a low level of prion seeding activity with no detectible protease-resistant PrP.

**Conclusions:** Cerebral organoid cultures can be used to model human prion disease *in vitro*. These structured 3D mini-tissues offer a capacity to investigate aspects of prion diseases never previously available in a human neuronal cell model. Potential human disease-specific applications include delineating sCJD subtype pathogenesis and testing putative therapeutics.

**KEYWORDS:** Cerebral organoid; sporadic CJD; induced pluripotent stem cell; RT-QuIC

References

[1] Krejciova Z, Alibhai J, Zhao C, et al. Human stem cell-derived astrocytes replicate human prions in a PRNP genotype-dependent manner. J Exp Med. 2017;214(12):3481-3495.

[2] Lancaster MA and Knoblich JA. Generation of cerebral organoids from human pluripotent stem cells. Nat Protoc. 2014;9(10):2329-2340.

[3] Renner M, Lancaster MA, Bian S, et al. Self-organized developmental patterning and differentiation in cerebral organoids. Embo J. 2017;36(10):1316-1329

**139. Cryo-electron microscopy of chronic wasting disease prions**

Sara Amidianª,ᵇ, Razieh Kamali-Jamilª,ᵇ, Ester Vázquez Fernándezª,ᵇ, Maria Carmen Garzaª,ᵇ,ᶜ, Camilo Duque Velásquezª,ᶜ, Xiongyao Wangª,ᵇ,ᶜ, Brian Tancownyª,ᵇ, Chiye Kimª, Judd Aikenª,ᵈ, Debbie McKenzieª,ᶜ, Howard Youngᵇ and Holger Willeª,ᵇ

CONTACT Sara Amidian [samidian@ualberta.ca](mailto:samidian@ualberta.ca)

Present address: ᵈUniversity of Zaragoza, Zaragoza, Aragón, Spain

Present address: ᶜSchool of Materials Science and Engineering, Harbin Institute of Technology, Weihai, Shandong, China.

ABSTRACT

**Introduction:** Chronic wasting disease (CWD) is a fatal neurodegenerative disease in cervids that belongs to a group of diseases known as prion diseases. CWD has been detected in Canada, United States, South Korea, Norway, and most recently in Finland. CWD is the most contagious prion disease and poses a risk for transmission to other species including humans.

**Aim:** PrP<sup>Sc</sup> is the misfolded form of the cellular prion protein (PrP<sup>C</sup>) and the infectious agent that causes prion diseases. The molecular mechanism of PrP<sup>C</sup> to PrP<sup>Sc</sup> transformation is unknown. To understand this structural conversion, it is essential to understand the structure of PrP<sup>Sc</sup>. Thus, the goal of this project is to investigate the structure of PrP<sup>Sc</sup> forms that are causing CWD, using negative stain and cryo electron microscopy (EM).

**Method:** PrP<sup>Sc</sup> amyloid fibrils were purified from the brains of CWD-infected Tg33 mice, which express deer prion

<footnote>ªLaboratory of Persistent Viral Diseases, National Institute of Allergy and Infectious Diseases, Division of Intramural Research, Rocky Mountain Laboratories, National Institutes of Health, 903 South 4th Street, Hamilton, MT 59,840; ᵇDepartment of Neurosciences, Biomedicine and Movement Sciences, University of Verona, Verona, Italy</footnote>

<footnote>ªCentre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Canada; ᵇDepartment of Biochemistry, University of Alberta, Edmonton, Canada; ᶜDepartment of Biological Sciences, University of Alberta, Edmonton, Canada; ᵈDepartment of Animal Health and Biomedical Sciences, University of Alberta, Edmonton, Canada</footnote>
<figure><img src="image_89.png" /></figure>

**138. Human cerebral organoids propagate sporadic CJD prions**

Bradley R. Grovemanª, Simote Foliakiª, Christina D. Orruª, Gianluigi Zanussoᵇ, James A. Carrollª, Brent Raceª and Kathryn L. Haighª

CONTACT Kathryn L. Haigh [cathryn.haigh@nih.gov](mailto:cathryn.haigh@nih.gov)

**ABSTRACT**

**Introduction:** Human prion disease has traditionally been difficult to model in cell culture. Very few human cell lines show sustained prion propagation over time and none represent three-dimensional (3D) brain tissue. Recently it was found that astrocyte cultures differentiated from human induced pluripotent stem cells (Hu-iPSCs) take up and propagate sporadic CJD (sCJD) prions with the resulting infection influenced by the sCJD subtype [1]. Hu-iPSCs have also been utilized to differentiate self-patterning, structured human brain tissues called cerebral organoids [2,3]. We hypothesized that these cerebral organoids could be used to model human prion propagation in vitro.

**Materials and Methods:** Cerebral organoid cultures were generated from hu-iPSCs using the protocol developed by Lancaster and Knoblich [2]. Cerebral organoids were infected with one of two sCJD inocula and monitored for health, morphology, PrP seeding activity, PrP deposition and proteinase-K resistant PrP as compared with organoids that received a normal brain homogenate inoculum.

**Results:** Cerebral organoids showed uptake and propagation of prions, detected by RT-QuIC analysis, following exposure to either sCJD inocula. Inoculum specific differences were observed. One inoculum showed robust proteinase-K resistant PrP in all tested organoids and coarse, granular PrP staining in the organoid interiors. The other inoculum induced a more toxic phenotype that affected organoid health but displayed a low level of prion seeding activity with no detectible protease-resistant PrP.

**Conclusions:** Cerebral organoid cultures can be used to model human prion disease *in vitro*. These structured 3D mini-tissues offer a capacity to investigate aspects of prion diseases never previously available in a human neuronal cell model. Potential human disease-specific applications include delineating sCJD subtype pathogenesis and testing putative therapeutics.

**KEYWORDS:** Cerebral organoid; sporadic CJD; induced pluripotent stem cell; RT-QuIC

**References**

[1] Krejciova Z, Alibhai J, Zhao C, et al. Human stem cell-derived astrocytes replicate human prions in a PRNP genotype-dependent manner. J Exp Med. 2017;214(12):3481–3495.

[2] Lancaster MA and Knoblich JA. Generation of cerebral organoids from human pluripotent stem cells. Nat Protoc. 2014;9(10):2329–2340.

[3] Renner M, Lancaster MA, Bian S, et al. Self-organized developmental patterning and differentiation in cerebral organoids. Embo J. 2017;36(10):1316–1329

**139. Cryo-electron microscopy of chronic wasting disease prions**

Sara Amidianª,ᵇ, Razieh Kamali-Jamilª,ᵇ, Ester Vázquez Fernándezª,ᵇ, Maria Carmen Garzaª,ᵇ,ᶜ, Camilo Duque Velásquezª,ᶜ, Xiongyao Wangª,ᵇ,ᶜ, Brian Tancownyª,ᵇ, Chiye Kimª, Judd Aikenª,ᵈ, Debbie McKenzieª,ᶜ, Howard Youngᵇ and Holger Willeª,ᵇ

CONTACT Sara Amidian [samidian@ualberta.ca](mailto:samidian@ualberta.ca)

Present address: ᵈUniversity of Zaragoza, Zaragoza, Aragón, Spain

Present address: ᶜSchool of Materials Science and Engineering, Harbin Institute of Technology, Weihai, Shandong, China.

**ABSTRACT**

**Introduction:** Chronic wasting disease (CWD) is a fatal neurodegenerative disease in cervids that belongs to a group of diseases known as prion diseases. CWD has been detected in Canada, United States, South Korea, Norway, and most recently in Finland. CWD is the most contagious prion disease and poses a risk for transmission to other species including humans.

**Aim:** PrP<sup>Sc</sup> is the misfolded form of the cellular prion protein (PrP<sup>C</sup>) and the infectious agent that causes prion diseases. The molecular mechanism of PrP<sup>C</sup> to PrP<sup>Sc</sup> transformation is unknown. To understand this structural conversion, it is essential to understand the structure of PrP<sup>Sc</sup>. Thus, the goal of this project is to investigate the structure of PrP<sup>Sc</sup> forms that are causing CWD, using negative stain and cryo electron microscopy (EM).

**Method:** PrP<sup>Sc</sup> amyloid fibrils were purified from the brains of CWD-infected Tg33 mice, which express deer prion

<footnote>ªLaboratory of Persistent Viral Diseases, National Institute of Allergy and Infectious Diseases, Division of Intramural Research, Rocky Mountain Laboratories, National Institutes of Health, 903 South 4th Street, Hamilton, MT 59,840; ᵇDepartment of Neurosciences, Biomedicine and Movement Sciences, University of Verona, Verona, Italy</footnote>

<footnote>ªCentre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Canada; ᵇDepartment of Biochemistry, University of Alberta, Edmonton, Canada; ᶜDepartment of Biological Sciences, University of Alberta, Edmonton, Canada; ᵈDepartment of Animal Health and Biomedical Sciences, University of Alberta, Edmonton, Canada</footnote>
<figure><img src="image_90.png" /></figure>

protein. To purify PrP<sup>Sc</sup> amyloid fibrils, phosphotungstate anions and sarkosyl were used. In addition, enzymes such as proteinase K (PK) or pronase E (PE) were added to remove PrP<sup>C</sup>. To assess the quality and quantity of the PrP<sup>Sc</sup> fibrils, the purified amyloid fibrils were negatively stained and visualized using EM. Cryo-EM and 3D fibril reconstruction are then performed on fibril preparations.

**Results and Conclusion:** PrP<sup>Sc</sup> amyloid fibrils were successfully purified and visualized using negative stain EM. Both, PK- and PE-purified samples displayed complex yet similar morphologies. However, a dominant and recognizable type seen in both preparations consisted of filaments with a clear twisted ribbon-like morphology. 2D class averages and 3D reconstructions from the negatively stained micrographs could help to better classify these fibrils. Additionally, cryo-EM analyses could resolve the structural differences between conformers of distinct CWD prion strains and their fibrillization properties. PE-purified fibrils, on the other hand, exhibited striations that run perpendicular to the fibril axis. Interestingly, the distance between these striations is ~40 nm, which corresponds to the molecular height of two PrP<sup>Sc</sup> molecules in a four-rung beta solenoid architecture that may adopt a head-to-head arrangement [1]. This observation could be explained by the presence of sarkosyl molecules associating with the hydrophobic GPI-anchor of the stacked PrP<sup>Sc</sup> molecules. Further image processing analyses will provide a more complete understanding of the orientation of the GPI-anchor in the CWD fibrils.

## References

[1] Wille H, Requena J. The Structure of PrP<sup>Sc</sup> Prions. Pathogens. 2018;7:20. DOI:10.3390/pathogens7010020.

# 140. Molecular dynamics simulations of cervid prion protein variants to assess protein stability and susceptibility towards chronic wasting disease

Sara Amidian<sup>a,b</sup>, Lyudmyla Dorosh<sup>c,d</sup>, Camilo Duque Velásquez<sup>a,e</sup>, Maria Stepanova<sup>c,d</sup>, Judd Aiken<sup>a,f</sup>, Debbie McKenzie<sup>a,e</sup> and Holger Wille<sup>a,b</sup>

CONTACT Sara Amidian samidian@ualberta.ca

## ABSTRACT

**Introduction:** Chronic wasting disease (CWD) is a prion disease that affects cervids. The central event in prion diseases is the structural conversion of the healthy, cellular prion protein (PrPC) to the disease-causing isoform, PrPSc. It is technically difficult to experimentally study the initial steps of the PrPC to PrPSc conversion; however, molecular dynamics (MD) simulation plays an important role in probing the misfolding events. One approach is to assess the effect of single-residue substitution on the structural stability of PrPC. Polymorphisms in the prion protein gene can influence prion disease susceptibility, disease progression, clinical presentation, and the propagation of distinct CWD strains associated with different PrPSc conformations. In this project, we use molecular dynamics simulation as a tool to characterize the effect of white-tailed deer (WTD) prion protein polymorphisms Q95H and G96S on the experimentally determined structure of the natively folded prion protein.

**Methods:** The initial model for the WTD prion protein (residues 93–233) was generated using the crystal structure of recombinant deer prion protein (PDB: 4YXH) as the template. The wild type (WT), 95H, and 96S models were subjected to minimizations, equilibrations, and production MD simulations using the Gromacs package. In total, three independent 50ns simulations were performed for each system.

**Results:** The 96S polymorphism displayed a higher root mean square deviation (RMSD) and root mean square fluctuation (RMSF) values, which are indicators of lower structural stability compared to WT and 95H forms. Also, 96S had a larger radius of gyration (Rg) values, indicating that the structure of 96S was less compactly folded than WT and 95H. Calculating the solvent accessible surface area (SASA) of 96S revealed that the structure of 96S was more exposed to the solvent. On the other hand, structural dynamics of the 95H polymorphism were closer to WT. RMSF and Rg values for 95H indicated higher stability and compactness of its structure. Additionally, the SASA values of 95H were very similar to WT.

**Conclusions:** Experimentally, the 96S allele is associated with slower disease progression and animals carrying this polymorphism have longer incubation time when inoculated with CWD prions. According to our MD results, it is apparent that the structure of WTD prion protein carrying the 96S polymorphism is less stable than the WT protein. The clear difference in the structural stability and local dynamics of 96S

<footnote><sup>a</sup>Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Canada; <sup>b</sup>Department of Biochemistry, University of Alberta, Edmonton, Canada; <sup>c</sup>National Institute for Nanotechnology, University of Alberta, Edmonton, Canada; <sup>d</sup>Department of Electrical and Computer Engineering, University of Alberta, Edmonton, Canada; <sup>e</sup>Department of Biological Sciences, University of Alberta, Edmonton, Canada; <sup>f</sup>Department of Animal Health and Biomedical Sciences, University of Alberta, Edmonton, Canada.</footnote>
<figure><img src="image_90.png" /></figure>

protein. To purify PrP<sup>Sc</sup> amyloid fibrils, phosphotungstate anions and sarkosyl were used. In addition, enzymes such as proteinase K (PK) or pronase E (PE) were added to remove PrP<sup>C</sup>. To assess the quality and quantity of the PrP<sup>Sc</sup> fibrils, the purified amyloid fibrils were negatively stained and visualized using EM. Cryo-EM and 3D fibril reconstruction are then performed on fibril preparations.

**Results and Conclusion:** PrP<sup>Sc</sup> amyloid fibrils were successfully purified and visualized using negative stain EM. Both, PK- and PE-purified samples displayed complex yet similar morphologies. However, a dominant and recognizable type seen in both preparations consisted of filaments with a clear twisted ribbon-like morphology. 2D class averages and 3D reconstructions from the negatively stained micrographs could help to better classify these fibrils. Additionally, cryo-EM analyses could resolve the structural differences between conformers of distinct CWD prion strains and their fibrillization properties. PE-purified fibrils, on the other hand, exhibited striations that run perpendicular to the fibril axis. Interestingly, the distance between these striations is ~40 nm, which corresponds to the molecular height of two PrP<sup>Sc</sup> molecules in a four-rung beta solenoid architecture that may adopt a head-to-head arrangement [1]. This observation could be explained by the presence of sarkosyl molecules associating with the hydrophobic GPI-anchor of the stacked PrP<sup>Sc</sup> molecules. Further image processing analyses will provide a more complete understanding of the orientation of the GPI-anchor in the CWD fibrils.

## References

[1] Wille H, Requena J. The Structure of PrP<sup>Sc</sup> Prions. Pathogens. 2018;7:20. DOI:10.3390/pathogens7010020.

# 140. Molecular dynamics simulations of cervid prion protein variants to assess protein stability and susceptibility towards chronic wasting disease

Sara Amidian<sup>a,b</sup>, Lyudmyla Dorosh<sup>c,d</sup>, Camilo Duque Velásquez<sup>a,e</sup>, Maria Stepanova<sup>c,d</sup>, Judd Aiken<sup>a,f</sup>, Debbie McKenzie<sup>a,e</sup> and Holger Wille<sup>a,b</sup>

CONTACT Sara Amidian samidian@ualberta.ca

## ABSTRACT

**Introduction:** Chronic wasting disease (CWD) is a prion disease that affects cervids. The central event in prion diseases is the structural conversion of the healthy, cellular prion protein (PrPC) to the disease-causing isoform, PrPSc. It is technically difficult to experimentally study the initial steps of the PrPC to PrPSc conversion; however, molecular dynamics (MD) simulation plays an important role in probing the misfolding events. One approach is to assess the effect of single-residue substitution on the structural stability of PrPC. Polymorphisms in the prion protein gene can influence prion disease susceptibility, disease progression, clinical presentation, and the propagation of distinct CWD strains associated with different PrPSc conformations. In this project, we use molecular dynamics simulation as a tool to characterize the effect of white-tailed deer (WTD) prion protein polymorphisms Q95H and G96S on the experimentally determined structure of the natively folded prion protein.

**Methods:** The initial model for the WTD prion protein (residues 93–233) was generated using the crystal structure of recombinant deer prion protein (PDB: 4YXH) as the template. The wild type (WT), 95H, and 96S models were subjected to minimizations, equilibrations, and production MD simulations using the Gromacs package. In total, three independent 50ns simulations were performed for each system.

**Results:** The 96S polymorphism displayed a higher root mean square deviation (RMSD) and root mean square fluctuation (RMSF) values, which are indicators of lower structural stability compared to WT and 95H forms. Also, 96S had a larger radius of gyration (Rg) values, indicating that the structure of 96S was less compactly folded than WT and 95H. Calculating the solvent accessible surface area (SASA) of 96S revealed that the structure of 96S was more exposed to the solvent. On the other hand, structural dynamics of the 95H polymorphism were closer to WT. RMSF and Rg values for 95H indicated higher stability and compactness of its structure. Additionally, the SASA values of 95H were very similar to WT.

**Conclusions:** Experimentally, the 96S allele is associated with slower disease progression and animals carrying this polymorphism have longer incubation time when inoculated with CWD prions. According to our MD results, it is apparent that the structure of WTD prion protein carrying the 96S polymorphism is less stable than the WT protein. The clear difference in the structural stability and local dynamics of 96S

<footnote><sup>a</sup>Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Canada; <sup>b</sup>Department of Biochemistry, University of Alberta, Edmonton, Canada; <sup>c</sup>National Institute for Nanotechnology, University of Alberta, Edmonton, Canada; <sup>d</sup>Department of Electrical and Computer Engineering, University of Alberta, Edmonton, Canada; <sup>e</sup>Department of Biological Sciences, University of Alberta, Edmonton, Canada; <sup>f</sup>Department of Animal Health and Biomedical Sciences, University of Alberta, Edmonton, Canada.</footnote>
<figure><img src="image_91.png" /></figure>

compared to WT, may be relevant to its partial resistance to prion disease, however, further analyses are required.

## 141. Propagation of human sCJD prions in organotypic slice culture

Grant Norman, Hailey Pineau and Valerie L. Sim

Department of Medicine, Division of Neurology, Faculty of Medicine & Dentistry, University of Alberta; Centre for Prions and Protein Folding Diseases, University of Alberta

CONTACT Valerie L. Sim valerie.sim@ualberta.ca

### ABSTRACT

Prion diseases, or transmissible spongiform encephalopathies (TSEs), are neurodegenerative diseases that are invariably fatal. These diseases result from the conversion of the normal prion protein (PrPC) to a misfolded form (PrPSc) that can template itself and spread through the brain, triggering neurodegeneration. TSEs can affect many mammals, including cervids (Chronic Wasting Disease; CWD), sheep (scrapie), cattle (Bovine Spongiform Encephalopathy; BSE), and humans (Creutzfeldt-Jakob Disease; CJD). There are no treatments for any of the prion diseases, despite decades of research. One challenge to finding successful therapies is the fact that PrPSc can exist in a number of different conformations, each of which may have its own bio-physical properties and cause distinct neuropathologies, giving rise to unique prion strain phenotypes. Treatments that target only one specific conformational change in may only work for one strain. Ideally, therapy studies for humans should be done with human prion strains. Here we demonstrate the propagation of human prions in prion organotypic slice culture with evidence of associated neuronal loss. Traditionally, prion strains are generated in animals infected with prion disease; however, these are lengthy experiments, often taking several months. By contrast, the prion organotypic slice culture assay (POSCA) can faithfully recapitulate aspects of prion pathology on a shortened timescale of 40–50 days [1,2]. This makes POSCA an ideal method for propagating prion strains. Originally, POSCA was developed to test mouse-adapted scrapie strains in cerebellar slices [2,3]. We have adapted POSCA to sporadic CJD strains from human samples and samples passaged through mice expressing human PrP. We have also adapted the system to whole brain coronal slices. The ability to propagate human prion strains ex vivo will allow investigation of human strain-specific properties during pathogenesis as well as development of strain-specific therapeutic interventions.

Because POSCA is an open system, it also allows for testing treatments at discrete disease time points without the confounder of blood-brain barrier.

### References

[1] Falsig J, et al. Nat Protoc. 2008;3(4):555–62.

[2] Falsig J, et al. PLoS Pathog. 2012;8(11):e1002985.

[3] Campeau J, et al. PLoS One. 2013;8(12):e81776.

## 142. Effects of Elk-PrP<sup>C</sup> expression levels on CWD strain properties

Camilo Duque Velásquez<sup>a</sup>, Alicia Otero<sup>a</sup>, Chiye Kim<sup>a</sup>, Elizabeth Triscott<sup>a</sup>, Jeffrey Narayan<sup>a</sup>, Jacques Van der Merwe<sup>b</sup>, Judd Aiken<sup>c</sup> and Debbie McKenzie<sup>a</sup>

CONTACT Alicia Otero oterogar@ualberta.ca

### ABSTRACT

Chronic Wasting Disease (CWD) is a contagious prion disease affecting various species of free-ranging and/or captive cervids on three continents. Species-specific prion protein (PrP<sup>C</sup>) polymorphisms influence prion conversion into PrP<sup>CWD</sup>. PrP<sup>C</sup> amino acid variation can also regulate disease susceptibility to particular prion strains and has been implicated in the diversification of prion strain conformers [1, 2, 3]. Elk and deer PrP<sup>C</sup> differ at residue E226Q and this amino acid difference has been implicated in the selection of CWD1 and CWD2 prion strains [4]. As PrP<sup>C</sup> expression has been suggested to affect prion strain evolution [5], we hypothesized that elk PrP<sup>C</sup> levels affect CWD strain generation. To test this hypothesis, transgenic (tg) FVB mice over-expressing elk PrP<sup>C</sup> [6] were crossed with *prnp* knock-out FVB mice to generate tg-elk with different PrP<sup>C</sup> expression levels. Both tg-elk<sup>+/+</sup> and tg-elk<sup>±</sup> were exposed to white-tailed deer CWD strains (Wisc-1 and H95<sup>+</sup>) [2, 3]. The H95<sup>+</sup> strain was a mixture generated on passage of Wisc-1 in deer heterozygous for H95G96 and Q95S96 [2]. Tg-elk<sup>+/+</sup> mice succumbed to Wisc-1 with a mean incubation period of 116 ± 7 days post infection (dpi) compared to 164 ± 11 dpi for the H95<sup>+</sup> strain mixture. Consistent with the reduced PrP<sup>C</sup> expression, the same deer prion strains resulted in longer incubation periods (157 ± 21 dpi and >180 dpi, respectively) when passaged in tg-elk<sup>±</sup> mice. After first passage, transmission of Wisc-1 and H95<sup>+</sup> in

<footnote><sup>a</sup>Department of Biological Sciences, Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Canada;</footnote>

<footnote><sup>b</sup>Department of Medicine, Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, AB, Canada;</footnote>

<footnote><sup>c</sup>Department of Agricultural, Food and Nutritional Sciences, Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, AB, Canada</footnote>
<figure><img src="image_91.png" /></figure>

compared to WT, may be relevant to its partial resistance to prion disease, however, further analyses are required.

## 141. Propagation of human sCJD prions in organotypic slice culture

Grant Norman, Hailey Pineau and Valerie L. Sim

Department of Medicine, Division of Neurology, Faculty of Medicine & Dentistry, University of Alberta; Centre for Prions and Protein Folding Diseases, University of Alberta

CONTACT Valerie L. Sim valerie.sim@ualberta.ca

### ABSTRACT

Prion diseases, or transmissible spongiform encephalopathies (TSEs), are neurodegenerative diseases that are invariably fatal. These diseases result from the conversion of the normal prion protein (PrPC) to a misfolded form (PrPSc) that can template itself and spread through the brain, triggering neurodegeneration. TSEs can affect many mammals, including cervids (Chronic Wasting Disease; CWD), sheep (scrapie), cattle (Bovine Spongiform Encephalopathy; BSE), and humans (Creutzfeldt-Jakob Disease; CJD). There are no treatments for any of the prion diseases, despite decades of research. One challenge to finding successful therapies is the fact that PrPSc can exist in a number of different conformations, each of which may have its own bio-physical properties and cause distinct neuropathologies, giving rise to unique prion strain phenotypes. Treatments that target only one specific conformational change in may only work for one strain. Ideally, therapy studies for humans should be done with human prion strains. Here we demonstrate the propagation of human prions in prion organotypic slice culture with evidence of associated neuronal loss. Traditionally, prion strains are generated in animals infected with prion disease; however, these are lengthy experiments, often taking several months. By contrast, the prion organotypic slice culture assay (POSCA) can faithfully recapitulate aspects of prion pathology on a shortened timescale of 40–50 days [1,2]. This makes POSCA an ideal method for propagating prion strains. Originally, POSCA was developed to test mouse-adapted scrapie strains in cerebellar slices [2,3]. We have adapted POSCA to sporadic CJD strains from human samples and samples passaged through mice expressing human PrP. We have also adapted the system to whole brain coronal slices. The ability to propagate human prion strains ex vivo will allow investigation of human strain-specific properties during pathogenesis as well as development of strain-specific therapeutic interventions.

Because POSCA is an open system, it also allows for testing treatments at discrete disease time points without the confounder of blood-brain barrier.

### References

[1] Falsig J, et al. Nat Protoc. 2008;3(4):555–62.

[2] Falsig J, et al. PLoS Pathog. 2012;8(11):e1002985.

[3] Campeau J, et al. PLoS One. 2013;8(12):e81776.

## 142. Effects of Elk-PrP<sup>C</sup> expression levels on CWD strain properties

Camilo Duque Velásquez<sup>a</sup>, Alicia Otero<sup>a</sup>, Chiye Kim<sup>a</sup>, Elizabeth Triscott<sup>a</sup>, Jeffrey Narayan<sup>a</sup>, Jacques Van der Merwe<sup>b</sup>, Judd Aiken<sup>c</sup> and Debbie McKenzie<sup>a</sup>

CONTACT Alicia Otero oterogar@ualberta.ca

### ABSTRACT

Chronic Wasting Disease (CWD) is a contagious prion disease affecting various species of free-ranging and/or captive cervids on three continents. Species-specific prion protein (PrP<sup>C</sup>) polymorphisms influence prion conversion into PrP<sup>CWD</sup>. PrP<sup>C</sup> amino acid variation can also regulate disease susceptibility to particular prion strains and has been implicated in the diversification of prion strain conformers [1, 2, 3]. Elk and deer PrP<sup>C</sup> differ at residue E226Q and this amino acid difference has been implicated in the selection of CWD1 and CWD2 prion strains [4]. As PrP<sup>C</sup> expression has been suggested to affect prion strain evolution [5], we hypothesized that elk PrP<sup>C</sup> levels affect CWD strain generation. To test this hypothesis, transgenic (tg) FVB mice over-expressing elk PrP<sup>C</sup> [6] were crossed with *prnp* knock-out FVB mice to generate tg-elk with different PrP<sup>C</sup> expression levels. Both tg-elk<sup>+/+</sup> and tg-elk<sup>±</sup> were exposed to white-tailed deer CWD strains (Wisc-1 and H95<sup>+</sup>) [2, 3]. The H95<sup>+</sup> strain was a mixture generated on passage of Wisc-1 in deer heterozygous for H95G96 and Q95S96 [2]. Tg-elk<sup>+/+</sup> mice succumbed to Wisc-1 with a mean incubation period of 116 ± 7 days post infection (dpi) compared to 164 ± 11 dpi for the H95<sup>+</sup> strain mixture. Consistent with the reduced PrP<sup>C</sup> expression, the same deer prion strains resulted in longer incubation periods (157 ± 21 dpi and >180 dpi, respectively) when passaged in tg-elk<sup>±</sup> mice. After first passage, transmission of Wisc-1 and H95<sup>+</sup> in

<footnote><sup>a</sup>Department of Biological Sciences, Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Canada;</footnote>

<footnote><sup>b</sup>Department of Medicine, Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, AB, Canada;</footnote>

<footnote><sup>c</sup>Department of Agricultural, Food and Nutritional Sciences, Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, AB, Canada</footnote>