<figure><img src="image_48.png" /></figure>

likely the best source of information about the potential for zoonotic transmission. The expected long incubation period, should transmission to humans occur, necessitates many years of vital status tracking.

**76. Prion disease incidence, United States, 2003–2016**

Ryan A. Maddox<sup>a</sup>, Marissa K. Person<sup>a</sup>, Janis E. Blevins<sup>b</sup>, Joseph Y. Abrams<sup>a</sup>, Bobbi L. Bryant<sup>a</sup>, Brian S. Appleby<sup>b</sup>, Lawrence B. Schonberger<sup>a</sup> and Ermias D. Belay<sup>a</sup>

<sup>a</sup>National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention (CDC), Atlanta, GA, USA; <sup>b</sup>National Prion Disease Pathology Surveillance Center (NPDPSC), Case Western Reserve University, Cleveland, OH, USA

CONTACT Ryan A. Maddox rmaddox@cdc.gov

**ABSTRACT**

**Introduction:** Mortality data and the results of neuro-pathological and genetic testing are used to estimate the incidence of prion diseases in the United States.

**Methods:** Prion disease decedents were identified from restricted-use U.S. national multiple cause-of-death data, via a data use agreement with the National Center for Health Statistics, and from the National Prion Disease Pathology Surveillance Center (NPDPSC) database for 2003–2016. NPDPSC decedents with neuropathological or genetic test results positive for prion disease for whom no likely match was found in the multiple cause-of-death data were added as cases for incidence calculations; those with negative neuropathology results but with cause-of-death data indicating prion disease were removed. Age-adjusted average annual incidence rates for the combined data were calculated using the year 2000 as the standard population.

**Results:** A total of 5737 decedents were identified as having prion disease during 2003–2016 for an age-adjusted average annual incidence of 1.2 per million populations. The age-adjusted annual incidence ranged from 1.0 per million in 2004 and 2006 to 1.4 per million in 2013; there was an increasing trend in age-adjusted incidence over the entire period (p <0.0001) but no significant increase during the second half (2010–2016, p = 0.08). The age-adjusted incidence between males and females (1.3 and 1.1 per million, respectively) differed significantly (p <0.0001). Twelve cases during 2003–2016 were <30 years of age for an age-specific incidence of 6.9 per billion; only two of these very young cases were sporadic, with the rest being familial (7), variant (2), or iatrogenic (1). The age-specific average annual incidence among those <55 and ≥55 during the time period was 0.2 and 4.7 per

million, respectively; incidence among those ≥65 was 5.9 per million.

**Conclusion:** The incidence of prion diseases, which are invariably fatal, can be estimated through analysis of mortality data supplemented with the results of neuro-pathological and genetic testing. Incidence in the United States over the last 7 years appears to be relatively stable. Cases <30 years of age continue to be very rare and usually indicate an exogenous source of infection or the presence of a genetic mutation.

**77. Assessing chronic wasting disease strain differences in free-ranging cervids across the United States**

Kaitlyn M. Wagner<sup>a</sup>, Caitlin Ott-Conn<sup>b</sup>, Kelly Straka<sup>b</sup>, Bob Dittmar<sup>c</sup>, Jasmine Batten<sup>d</sup>, Robyn Pierce<sup>a</sup>, Mercedes Hennessy<sup>a</sup>, Elizabeth Gordon<sup>a</sup>, Brett Israel<sup>a</sup>, Jenn Ballard<sup>e</sup> and Mark D Zabel<sup>a</sup>

<sup>a</sup>Prion Research Center at Colorado State University; <sup>b</sup>Michigan Department of Natural Resources; <sup>c</sup>Texas Parks and Wildlife Department; <sup>d</sup>Missouri Department of Conservation, 5. Arkansas Game and Fish Commission

CONTACT Kaitlyn M. Wagner miedkait@rams.colostate.edu

**ABSTRACT**

**Background/Introduction:** Chronic wasting disease (CWD) is an invariably fatal prion disease affecting captive and free-ranging cervids, including white-tailed deer, mule deer, moose, elk, and reindeer. Since the initial description of the disease in the 1960's, CWD has spread to 23 states, 3 Canadian Provinces, South Korea, Norway and, most recently, Finland. While some outbreaks of CWD were caused by transport of infected animals from endemic regions, the origin of CWD in other epizootics is unclear and has not been characterized. Previous studies have shown that there are two distinct strains of CWD. However, the continuous spread and the unclear origin of several outbreaks warrant continued surveillance and further characterization of strain diversity.

**Materials and Methods:** To address these knowledge gaps, we used biochemical tests to assess strain differences between CWD outbreaks in Michigan, Texas, Missouri, and Colorado, USA. Brain or lymph node samples were homogenized and digested in 50 µg/mL proteinase K (PK). These samples were then run on a Western blot to assess glycoform ratio and electrophoretic mobility. Texas samples were digested in 100 µg/mL PK. To assess conformational stability, brain or lymph node homogenates were incubated in increasing concentrations of guanidine hydrochloride from 0 M to 4 M in 0.5 M increments. Samples were
<figure><img src="image_52.png" /></figure>

likely the best source of information about the potential for zoonotic transmission. The expected long incubation period, should transmission to humans occur, necessitates many years of vital status tracking.

**76. Prion disease incidence, United States, 2003–2016**

Ryan A. Maddox<sup>a</sup>, Marissa K. Person<sup>a</sup>, Janis E. Blevins<sup>b</sup>, Joseph Y. Abrams<sup>a</sup>, Bobbi L. Bryant<sup>a</sup>, Brian S. Appleby<sup>b</sup>, Lawrence B. Schonberger<sup>a</sup> and Ermias D. Belay<sup>a</sup>

<sup>a</sup>National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention (CDC), Atlanta, GA, USA; <sup>b</sup>National Prion Disease Pathology Surveillance Center (NPDPSC), Case Western Reserve University, Cleveland, OH, USA

CONTACT Ryan A. Maddox rmaddox@cdc.gov

**ABSTRACT**

**Introduction:** Mortality data and the results of neuro-pathological and genetic testing are used to estimate the incidence of prion diseases in the United States.

**Methods:** Prion disease decedents were identified from restricted-use U.S. national multiple cause-of-death data, via a data use agreement with the National Center for Health Statistics, and from the National Prion Disease Pathology Surveillance Center (NPDPSC) database for 2003–2016. NPDPSC decedents with neuropathological or genetic test results positive for prion disease for whom no likely match was found in the multiple cause-of-death data were added as cases for incidence calculations; those with negative neuropathology results but with cause-of-death data indicating prion disease were removed. Age-adjusted average annual incidence rates for the combined data were calculated using the year 2000 as the standard population.

**Results:** A total of 5737 decedents were identified as having prion disease during 2003–2016 for an age-adjusted average annual incidence of 1.2 per million populations. The age-adjusted annual incidence ranged from 1.0 per million in 2004 and 2006 to 1.4 per million in 2013; there was an increasing trend in age-adjusted incidence over the entire period (p <0.0001) but no significant increase during the second half (2010–2016, p = 0.08). The age-adjusted incidence between males and females (1.3 and 1.1 per million, respectively) differed significantly (p <0.0001). Twelve cases during 2003–2016 were <30 years of age for an age-specific incidence of 6.9 per billion; only two of these very young cases were sporadic, with the rest being familial (7), variant (2), or iatrogenic (1). The age-specific average annual incidence among those <55 and ≥55 during the time period was 0.2 and 4.7 per

million, respectively; incidence among those ≥65 was 5.9 per million.

**Conclusion:** The incidence of prion diseases, which are invariably fatal, can be estimated through analysis of mortality data supplemented with the results of neuro-pathological and genetic testing. Incidence in the United States over the last 7 years appears to be relatively stable. Cases <30 years of age continue to be very rare and usually indicate an exogenous source of infection or the presence of a genetic mutation.

**77. Assessing chronic wasting disease strain differences in free-ranging cervids across the United States**

Kaitlyn M. Wagner<sup>a</sup>, Caitlin Ott-Conn<sup>b</sup>, Kelly Straka<sup>b</sup>, Bob Dittmar<sup>c</sup>, Jasmine Batten<sup>d</sup>, Robyn Pierce<sup>a</sup>, Mercedes Hennessy<sup>a</sup>, Elizabeth Gordon<sup>a</sup>, Brett Israel<sup>a</sup>, Jenn Ballard<sup>e</sup> and Mark D Zabel<sup>a</sup>

<sup>a</sup>Prion Research Center at Colorado State University; <sup>b</sup>Michigan Department of Natural Resources; <sup>c</sup>Texas Parks and Wildlife Department; <sup>d</sup>Missouri Department of Conservation, 5. Arkansas Game and Fish Commission

CONTACT Kaitlyn M. Wagner miedkait@rams.colostate.edu

**ABSTRACT**

**Background/Introduction:** Chronic wasting disease (CWD) is an invariably fatal prion disease affecting captive and free-ranging cervids, including white-tailed deer, mule deer, moose, elk, and reindeer. Since the initial description of the disease in the 1960's, CWD has spread to 23 states, 3 Canadian Provinces, South Korea, Norway and, most recently, Finland. While some outbreaks of CWD were caused by transport of infected animals from endemic regions, the origin of CWD in other epizootics is unclear and has not been characterized. Previous studies have shown that there are two distinct strains of CWD. However, the continuous spread and the unclear origin of several outbreaks warrant continued surveillance and further characterization of strain diversity.

**Materials and Methods:** To address these knowledge gaps, we used biochemical tests to assess strain differences between CWD outbreaks in Michigan, Texas, Missouri, and Colorado, USA. Brain or lymph node samples were homogenized and digested in 50 µg/mL proteinase K (PK). These samples were then run on a Western blot to assess glycoform ratio and electrophoretic mobility. Texas samples were digested in 100 µg/mL PK. To assess conformational stability, brain or lymph node homogenates were incubated in increasing concentrations of guanidine hydrochloride from 0 M to 4 M in 0.5 M increments. Samples were
<figure><img src="image_49.png" /></figure>

then precipitated in methanol overnight, washed and
PK digested in 50 µg/mL PK before slot blotting.

**Results:** Our results have found significant differences in glycoform ratio between CWD from Michigan and Colorado, but no differences were observed in conformational stability assays. Interestingly, when testing our CWD isolates from Texas to analyse electrophoretic mobility and glycoform ratio, we found that these samples did not exhibit the characteristic band shift when treated with PK, but PK resistant material remained. Additionally, results from our conformational stability assay demonstrate a unique profile of these Texas isolates. Testing of samples from Missouri is currently underway.

**Conclusions:** Thus far, our data indicate that there are strain differences between CWD circulating in Michigan and CWD in Colorado and provide important insight into CWD strain differences between two non-contiguous outbreaks. We have also identified a unique strain of CWD in Texas with biochemical strain properties not seen in any of our other CWD isolates. These results highlight the importance of continued surveillance to better understand this devastating disease. These results have important implications for CWD emergence, evolution and our understanding of prion strain heterogeneity on the landscape.

**78. Optogenetically stimulation of medial septum-diagonal band complex ameliorates the cognitive dysfunctions of APP<sup>NL-G-F</sup>, mouse model of Alzheimer's disease**

Jogender Mehla, Surjeet Singh, Robert J. McDonald and Majid H. Mohajerani

Canadian Centre for Behavioural Neuroscience, University of Lethbridge, Lethbridge, Alberta, Canada.

CONTACT Jogender Mehla jogender.mehla@uleth.cal; jsmeh-laaiims@gmail.com

**ABSTRACT**

**Introduction:** Cholinergic neuron degeneration in the basal forebrain is known to be one of the initial pathological hallmarks of Alzheimer's disease (AD). Additionally, currently available drug therapy is also based on the cholinergic hypothesis which provides only symptomatic treatment. Therefore, considering the importance of cholinergic system in AD, we planned this study to investigate the effect of chronic optogenetic stimulation of medial septum-diagonal band (MSDB) complex, a part of basal forebrain on the cognitive deficits and amyloid pathology in knock-in mouse model of AD.

**Materials and Methods:** In the present study, we used male APP<sup>NL-G-F</sup> mice. We used male C57BL/6J mice as a normal control. We injected channelrhodopsin virus and

implanted optrodes into MSDB complex of 2 months old APP<sup>NL-G-F</sup> mice. After one month of surgery, these mice were divided into two groups; one, where no optogenetic stimulation was done and second, in which MSDB stimulation was performed for 3 months using blue light (473 nm, 3–4 mW). After end of 3 months stimulation, Morris water maze (MWM) and fear conditioning tests were performed for the assessment of learning and memory functions. Lastly, we perfused the mice and brains were extracted for immunostaining.

**Results:** The results show that optogenetically stimulation of MSDB improved the learning ability of APP<sup>NL-G-F</sup> mice in MWM evidenced by significant decrease in escape latency on day 8 in comparison with APP<sup>NL-G-F</sup> sham group. Furthermore, optogenetic stimulated mice spent significantly more time in target quadrant as compared to average of other quadrants. Nonetheless, optogenetically stimulated mice also spent significantly more time in target quadrant in comparison with APP<sup>NL-G-F</sup> sham group indicating an improvement in memory retention. APP<sup>NL-G-F</sup> mice also showed an improvement in memory function in tone and contextual fear conditioning tests. The chronic optogenetically stimulation of MSDB reduced the amyloid burden in cortex and hippocampus of APP<sup>NL-G-F</sup> mice which may be responsible for improvement in cognitive functions.

**Conclusion:** The findings from the present study support the potential of cholinergic hypothesis and optogenetics for the treatment of AD.

**79. The FXR1 protein is a functional amyloid of mammalian brain**

A. V. Sergeevaª, M. E. Velizhaninaª, J. V. Sopovaª,b, E. I. Koshelᶜ, T. A. Belashovaᵇ, S. P. Zadorskyª,b, V. A. Siniukovaᵇ and A. P. Galkinª,b

ªDepartment of Genetics and Biotechnology, St. Petersburg State University, Petersburg, Russian Federation; ᵇVavilov Institute of General Genetics, St. Petersburg Branch, Russian Academy of Sciences, Petersburg, Russian Federation; ᶜDepartment of Cytology and Histology, St. Petersburg State University, St. Petersburg, Russian Federation

CONTACT A. V. Sergeeva Aleksandra.helwig@yandex.ru

Amyloids are non-branching fibrils that are composed of stacked monomers stabilized by intermolecular β-sheets. Our novel proteomic approach PSIA-LC-MALDI separates amyloids from the majority of other protein complexes of a non-amyloid nature. Being applied to *R. norvegicus* brain proteome, PSIA-LC-MALDI allowed identification of several amyloid-like proteins forming SDS-resistant aggregates. FXR1 was among identified proteins. FXR1 binds and regulates translation of target mRNAs and takes part in memory and emotions control.
<figure><img src="image_53.png" /></figure>

then precipitated in methanol overnight, washed and
PK digested in 50 µg/mL PK before slot blotting.

**Results:** Our results have found significant differences in glycoform ratio between CWD from Michigan and Colorado, but no differences were observed in conformational stability assays. Interestingly, when testing our CWD isolates from Texas to analyse electrophoretic mobility and glycoform ratio, we found that these samples did not exhibit the characteristic band shift when treated with PK, but PK resistant material remained. Additionally, results from our conformational stability assay demonstrate a unique profile of these Texas isolates. Testing of samples from Missouri is currently underway.

**Conclusions:** Thus far, our data indicate that there are strain differences between CWD circulating in Michigan and CWD in Colorado and provide important insight into CWD strain differences between two non-contiguous outbreaks. We have also identified a unique strain of CWD in Texas with biochemical strain properties not seen in any of our other CWD isolates. These results highlight the importance of continued surveillance to better understand this devastating disease. These results have important implications for CWD emergence, evolution and our understanding of prion strain heterogeneity on the landscape.

**78. Optogenetically stimulation of medial septum-diagonal band complex ameliorates the cognitive dysfunctions of APP<sup>NL-G-F</sup>, mouse model of Alzheimer's disease**

Jogender Mehla, Surjeet Singh, Robert J. McDonald and Majid H. Mohajerani

Canadian Centre for Behavioural Neuroscience, University of Lethbridge, Lethbridge, Alberta, Canada.

**CONTACT** Jogender Mehla jogender.mehla@uleth.cal; jsmeh-laaiims@gmail.com

**ABSTRACT**

**Introduction:** Cholinergic neuron degeneration in the basal forebrain is known to be one of the initial pathological hallmarks of Alzheimer's disease (AD). Additionally, currently available drug therapy is also based on the cholinergic hypothesis which provides only symptomatic treatment. Therefore, considering the importance of cholinergic system in AD, we planned this study to investigate the effect of chronic optogenetic stimulation of medial septum-diagonal band (MSDB) complex, a part of basal forebrain on the cognitive deficits and amyloid pathology in knock-in mouse model of AD.

**Materials and Methods:** In the present study, we used male APP<sup>NL-G-F</sup> mice. We used male C57BL/6J mice as a normal control. We injected channelrhodopsin virus and

implanted optrodes into MSDB complex of 2 months old APP<sup>NL-G-F</sup> mice. After one month of surgery, these mice were divided into two groups; one, where no optogenetic stimulation was done and second, in which MSDB stimulation was performed for 3 months using blue light (473 nm, 3–4 mW). After end of 3 months stimulation, Morris water maze (MWM) and fear conditioning tests were performed for the assessment of learning and memory functions. Lastly, we perfused the mice and brains were extracted for immunostaining.

**Results:** The results show that optogenetically stimulation of MSDB improved the learning ability of APP<sup>NL-G-F</sup> mice in MWM evidenced by significant decrease in escape latency on day 8 in comparison with APP<sup>NL-G-F</sup> sham group. Furthermore, optogenetic stimulated mice spent significantly more time in target quadrant as compared to average of other quadrants. Nonetheless, optogenetically stimulated mice also spent significantly more time in target quadrant in comparison with APP<sup>NL-G-F</sup> sham group indicating an improvement in memory retention. APP<sup>NL-G-F</sup> mice also showed an improvement in memory function in tone and contextual fear conditioning tests. The chronic optogenetically stimulation of MSDB reduced the amyloid burden in cortex and hippocampus of APP<sup>NL-G-F</sup> mice which may be responsible for improvement in cognitive functions.

**Conclusion:** The findings from the present study support the potential of cholinergic hypothesis and optogenetics for the treatment of AD.

**79. The FXR1 protein is a functional amyloid of mammalian brain**

A. V. Sergeevaª, M. E. Velizhaninaª, J. V. Sopovaª,ᵇ, E. I. Koshelᶜ, T. A. Belashovaᵇ, S. P. Zadorskyª,ᵇ, V. A. Siniukovaᵇ and A. P. Galkinª,ᵇ

ªDepartment of Genetics and Biotechnology, St. Petersburg State University, Petersburg, Russian Federation; ᵇVavilov Institute of General Genetics, St. Petersburg Branch, Russian Academy of Sciences, Petersburg, Russian Federation; ᶜDepartment of Cytology and Histology, St. Petersburg State University, St. Petersburg, Russian Federation

**CONTACT** A. V. Sergeeva Aleksandra.helwig@yandex.ru

Amyloids are non-branching fibrils that are composed of stacked monomers stabilized by intermolecular β-sheets. Our novel proteomic approach PSIA-LC-MALDI separates amyloids from the majority of other protein complexes of a non-amyloid nature. Being applied to *R. norvegicus* brain proteome, PSIA-LC-MALDI allowed identification of several amyloid-like proteins forming SDS-resistant aggregates. FXR1 was among identified proteins. FXR1 binds and regulates translation of target mRNAs and takes part in memory and emotions control.
<figure><img src="image_50.png" /></figure>

We demonstrated that FXR1 forms amyloid conformers that strongly co-localize with thioflavin S, bind RNA and prevent its RNAseA-mediated degradation in the cortex neurons of the rat brain. Furthermore, we showed that N-terminal region of the FXR1 protein underlies the ability of full-length FXR1 to form SDS-resistant oligomers and insoluble aggregates in the brains of young and healthy rat males and forms amyloid fibrils *in vitro*. Moreover, *in vitro* assay confirmed the ability of amyloid conformers of the N-terminal region of FXR1 to bind poly(A)-RNA extracted from the rat brain. To further evaluate the amyloidogenic properties of FXR1 N-terminal region we used bacterial-based C-DAG system. Not to our surprise, FXR1(1–380) forms extracellular fibrils in bacteria, that can be visualized by TEM and demonstrates apple-green birefringence when stained with Congo Red and viewed under crossed polarizers. Specification of FXR1's amyloidogenic region boundaries allowed us to shorten it from (aa 1–380) to (aa 1–270), as FXR1(1–270) demonstrates same amyloidogenic properties as FXR1(1–380) in C-DAG system. To shed the light on the possibility of the FXR1 protein being a functional amyloid of human brain as well, we used a human neuroblastoma cell line, IMR32, as a human brain model. We demonstrated that in human neuroblastoma cells FXR1 forms insoluble aggregates of high molecular mass the abundance of which is controlled in serum-dependent manner. Considering the fact that serum starvation is one of the standard methods of inducing neuroblastoma cells differentiation, we speculate that FXR1 oligomers and amyloid aggregates may play a role in cell differentiation via its interaction with mRNAs of cell cycle proteins, such as p21. Interestingly, the N-terminal region of FXR1 is highly conserved across mammals and contains identically arranged potentially amyloidogenic sequences in evolutionary distinct vertebrates. These data support our hypothesis that the ability of FXR1 to function in amyloid form is conserved in evolution. The reported study was funded by RFBR according to the research project 18–34-00420 and grant of SpbSU to Laboratory of Amyloid Biology.

**80. Cellular prion protein promotes cell metabolic orientation towards the oxidative degradation of glucose**

H. Arnouldª, A. Baudryª, M. Pietriª, A. M. Haeberleᵇ, M. Laforgeª, G. Berthoᶜ, G. Schmitt-Ulmsᵈ, Y. Baillyᵇ, O. Kellermannª and B. Schneiderª

CONTACT H. Arnould helene.arnould@parisdescartes.fr

While the implication of cellular prion protein PrP<sup>C</sup> has been widely documented in prion diseases, the role(s) of this ubiquitous protein still remain(s) elusive. To address this issue, PrP<sup>C</sup> was chronically silenced in the 1C11 neuronal cell line, a neuronal stem cell endowed with the capacity to differentiate into serotonergic or noradrenergic neurons. A global analysis was performed to compare the proteome of PrP<sup>C</sup>-depleted 1C11 cells (referred to as PrPnull-1C11 cells) to that of parental PrP<sup>C</sup>-expressing 1C11 cells with the aim to identify cell function(s) deregulated by the absence of PrP<sup>C</sup>. Our proteomic analysis reveals strong abnormalities in the expression level of enzymes at play in the glucose energetic metabolism in PrPnull-1C11 cells with a reduced expression of mitochondrial enzymes in favour of the enzymes involved in the glycolytic-fermentative pathway. Surprisingly, functional experiments by NMR and Seahorse approaches to probe metabolic fluxes in cells indicate reduced glycolytic and lactic fermentative fluxes as well as decreased mitochondrial oxygen consumption in PrPnull-1C11 cells when grown in a medium containing glucose. Global reduction of glucose oxidative degradation in PrP<sup>C</sup>-depleted cells is associated with down-expression of the glucose transporter GLUT3 and down-regulation of pyruvate dehydrogenase (PDHA1) activity that normally promotes the entry of pyruvate into mitochondria and transformation into acetyl-CoA. Finally, we show a metabolic shift in PrPnull-1C11 cells with a preferred orientation of the energetic metabolism towards fatty acid degradation by the β-oxidation pathway to maintain ATP concentration at high levels. The flip side of such conversion in the energetic pathways is the onset of oxidative stress conditions in PrP<sup>C</sup>-depleted cells. For the first time, our work provides evidence that PrP<sup>C</sup> plays a key role in the energetic metabolism by potentiating glucose oxidative degradation through the glycolysis-mitochondria pathway. As corruption of PrP<sup>C</sup> function(s) by pathogenic prions is at the root of prion diseases, any perturbation of PrP<sup>C</sup> control of glucose degradation in prion-infected cells may contribute to neurodegeneration.

**KEYWORDS:** Proteomics; PrP<sup>C</sup> function; glucose and fatty acid energetic metabolisms; oxidative stress

**81. Detection of neuronal dysfunction associated with genetic prion disease in human cerebral organoids**

Simote T. Foliakiª, Bradley R. Grovemanª, Aleksandar R. Woodª, Jue Yuanᵇ, Leslie Coopermanᵇ, Paul Tesarᵇ, Wen-Quan Zouᵇ and Kathryn L. Haighª

<footnote>ªINSERM UMR 1124, Université Paris Descartes, Paris, France; ᵇCNRS UPR 3212, Institut des Neurosciences Cellulaires et Intégratives, Strasbourg, France; ᶜCNRS UMR 8601, Université Paris Descartes, Paris, France; ᵈTanz Centre for Research in Neurodegenerative Diseases, University of Toronto, Canada</footnote>
<figure><img src="image_54.png" /></figure>

We demonstrated that FXR1 forms amyloid conformers that strongly co-localize with thioflavin S, bind RNA and prevent its RNAseA-mediated degradation in the cortex neurons of the rat brain. Furthermore, we showed that N-terminal region of the FXR1 protein underlies the ability of full-length FXR1 to form SDS-resistant oligomers and insoluble aggregates in the brains of young and healthy rat males and forms amyloid fibrils *in vitro*. Moreover, *in vitro* assay confirmed the ability of amyloid conformers of the N-terminal region of FXR1 to bind poly(A)-RNA extracted from the rat brain. To further evaluate the amyloidogenic properties of FXR1 N-terminal region we used bacterial-based C-DAG system. Not to our surprise, FXR1(1–380) forms extracellular fibrils in bacteria, that can be visualized by TEM and demonstrates apple-green birefringence when stained with Congo Red and viewed under crossed polarizers. Specification of FXR1's amyloidogenic region boundaries allowed us to shorten it from (aa 1–380) to (aa 1–270), as FXR1(1–270) demonstrates same amyloidogenic properties as FXR1(1–380) in C-DAG system. To shed the light on the possibility of the FXR1 protein being a functional amyloid of human brain as well, we used a human neuroblastoma cell line, IMR32, as a human brain model. We demonstrated that in human neuroblastoma cells FXR1 forms insoluble aggregates of high molecular mass the abundance of which is controlled in serum-dependent manner. Considering the fact that serum starvation is one of the standard methods of inducing neuroblastoma cells differentiation, we speculate that FXR1 oligomers and amyloid aggregates may play a role in cell differentiation via its interaction with mRNAs of cell cycle proteins, such as p21. Interestingly, the N-terminal region of FXR1 is highly conserved across mammals and contains identically arranged potentially amyloidogenic sequences in evolutionary distinct vertebrates. These data support our hypothesis that the ability of FXR1 to function in amyloid form is conserved in evolution. The reported study was funded by RFBR according to the research project 18–34-00420 and grant of SpbSU to Laboratory of Amyloid Biology.

**80. Cellular prion protein promotes cell metabolic orientation towards the oxidative degradation of glucose**

H. Arnould<sup>a</sup>, A. Baudry<sup>a</sup>, M. Pietri<sup>a</sup>, A. M. Haeberle<sup>b</sup>, M. Laforge<sup>a</sup>, G. Bertho<sup>c</sup>, G. Schmitt-Ulms<sup>d</sup>, Y. Bailly<sup>b</sup>, O. Kellermann<sup>a</sup> and B. Schneider<sup>a</sup>

CONTACT H. Arnould helene.arnould@parisdescartes.fr

While the implication of cellular prion protein $PrP^C$ has been widely documented in prion diseases, the role(s) of this ubiquitous protein still remain(s) elusive. To address this issue, $PrP^C$ was chronically silenced in the 1C11 neuronal cell line, a neuronal stem cell endowed with the capacity to differentiate into serotonergic or noradrenergic neurons. A global analysis was performed to compare the proteome of $PrP^C$-depleted 1C11 cells (referred to as PrPnull-1C11 cells) to that of parental $PrP^C$-expressing 1C11 cells with the aim to identify cell function(s) deregulated by the absence of $PrP^C$. Our proteomic analysis reveals strong abnormalities in the expression level of enzymes at play in the glucose energetic metabolism in PrPnull-1C11 cells with a reduced expression of mitochondrial enzymes in favour of the enzymes involved in the glycolytic-fermentative pathway. Surprisingly, functional experiments by NMR and Seahorse approaches to probe metabolic fluxes in cells indicate reduced glycolytic and lactic fermentative fluxes as well as decreased mitochondrial oxygen consumption in PrPnull-1C11 cells when grown in a medium containing glucose. Global reduction of glucose oxidative degradation in $PrP^C$-depleted cells is associated with down-expression of the glucose transporter GLUT3 and down-regulation of pyruvate dehydrogenase (PDHA1) activity that normally promotes the entry of pyruvate into mitochondria and transformation into acetyl-CoA. Finally, we show a metabolic shift in PrPnull-1C11 cells with a preferred orientation of the energetic metabolism towards fatty acid degradation by the β-oxidation pathway to maintain ATP concentration at high levels. The flip side of such conversion in the energetic pathways is the onset of oxidative stress conditions in $PrP^C$-depleted cells. For the first time, our work provides evidence that $PrP^C$ plays a key role in the energetic metabolism by potentiating glucose oxidative degradation through the glycolysis-mitochondria pathway. As corruption of $PrP^C$ function(s) by pathogenic prions is at the root of prion diseases, any perturbation of $PrP^C$ control of glucose degradation in prion-infected cells may contribute to neurodegeneration.

**KEYWORDS:** Proteomics; $PrP^C$ function; glucose and fatty acid energetic metabolisms; oxidative stress

**81. Detection of neuronal dysfunction associated with genetic prion disease in human cerebral organoids**

Simote T. Foliaki<sup>a</sup>, Bradley R. Groveman<sup>a</sup>, Aleksandar R. Wood<sup>a</sup>, Jue Yuan<sup>b</sup>, Leslie Cooperman<sup>b</sup>, Paul Tesar<sup>b</sup>, Wen-Quan Zou<sup>b</sup> and Kathryn L. Haigh<sup>a</sup>

<footnote><sup>a</sup>INSERM UMR 1124, Université Paris Descartes, Paris, France; <sup>b</sup>CNRS UPR 3212, Institut des Neurosciences Cellulaires et Intégratives, Strasbourg, France; <sup>c</sup>CNRS UMR 8601, Université Paris Descartes, Paris, France; <sup>d</sup>Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, Canada</footnote>
<figure><img src="image_51.png" /></figure>

CONTACT Simote T. Foliaki simote.foliaki@nih.gov

## ABSTRACT

**Introduction:** One of the fundamental problems of researching human prion diseases is access to live human brain tissue. Utilizing stem cell technology to produce human cerebral organoids (COs), which currently represent the nearest model to structured human brain tissue, offers a new avenue of investigation [1]. These organoids are self-organizing, structured regions analogous to human brain tissue that mature to contain multiple neuronal populations as well as astrocytes and oligodendrocytes [1, 2]. Importantly, these COs could be generated from donors who are carriers of mutations associated with familial prion diseases such as the E200K mutation. Herein we aimed to investigate the influence of E200K mutation that pre-dispose individuals to prion disease on neuronal electrical signalling.

**Methods:** COs have been generated from donors with E200K mutations (pre-disposed) and without (non-predisposed) mutations within the prion gene using the protocol described by Lancaster and Knoblick [1]. To measure the ability of these organoids to evoke spontaneous neuronal electrical signalling, they were sliced into 300 µm thick slices and mounted onto multi-electrode arrays (MEAs). MEAs are embedded with micro-electrodes to measure extracellular neuronal electrical spike (action potentials) and network communication throughout individual organoid. During the recording, the organoid slices were provided with adequate nutrients and oxygen through continuous superfusion with oxygenated artificial cerebrospinal fluid. To induce electrical signalling associated with cognition and behaviour, we exposed the slices to known stimulants and depressants of neuronal activity to determine their ability to express functions associated with memory formation and those related to memory loss [3, 4].

**Results and Conclusion:** We found that both non-predisposed and predisposed organoids exhibited active spontaneous neuronal function and network activity. Exposure of these organoids to glycine induced a persistently enhanced rate of neuronal spikes, a physiological correlate of memory formation called Long-Term Potentiation (LTP). Exposure of these organoids to glutamate/glycine induced LTP as well as Long-Term Depression (LTD), a physiological correlate of memory loss. Overall, we observed differences between the predisposed and non-predisposed organoids in neuronal excitability and response to depressants which

may influence excitotoxicity events and level of fatigue in neuronal activity in these organoids.

## References

[1] Lancaster, et al. Nat Protoc. 2014;9(10):2329-2340.

[2] Renner, et al. Embo J. 2017;36(10):1316-1329.

[3] Igartua, et al. Neuropharmacology. 2007;52(8):1586-1595.

[4] Hasegawa, et al. Sci Rep. 2015;5:7707.

## 82. The celecoxib derivative AR-12 induces autophagy and controls prion infection in vitro and in vivo

Basant A. Abdulrahmanª,ᵇ, Dalia Abdelazizª,ᶜ, Simrika Thapaª,ᶜ, Li Luª,ᶜ, Sabine Gilchᵇ,ᶜ and Hermann M. SchatzIª,ᶜ

CONATCT Basant A. Abdulrahman baabdulr@ucalgary.ca

## ABSTRACT

Prion diseases are fatal infectious neurodegenerative disorders that affect both humans and animals. The autocatalytic conversion of the cellular prion protein (PrPC) into the pathologic isoform PrPSc is a key feature in prion pathogenesis. AR-12 is an IND-approved derivative of celecoxib that demonstrated preclinical activity against several microbial diseases. Loss of neurons, astrogliosis and mild microglia activation are the main pathological features of prion diseases. This results in a progressive spongiform degeneration of the central nervous system (CNS), leading to ataxia, behavioural changes and, in humans, highly progressive loss of intellectual abilities. In the last two decades, great efforts have been made to establish treatment options for prion diseases. These included testing existing drugs for anti-prion activity in experimental models with only a few agents progressing to human studies of patients with prion diseases. Investigations to date have not resulted in a recognized/proven treatment for prion diseases. Recently, AR-12 (a.k.a. OSU-03012), a potent inhibitor of PDK-1 (phosphoinositide-dependent kinase 1) and autophagy stimulator, has been shown to facilitate clearance of misfolded proteins. The latter proposes AR-12 to be a

<footnote>ªNational Institute of Allergy and Infectious Diseases, NIH; ᵇCase Western Reserve University School of Medicine</footnote>

<footnote>ªDepartment of Comparative Biology & Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, Canada; ᵇDepartment of Ecosystem & Public Health, Faculty of Veterinary Medicine, University of Calgary, Calgary, Canada; ᶜCalgary Prion Research Unit, and Hotchkiss Brain Institute, University of Calgary, Calgary, Canada</footnote>
<figure><img src="image_55.png" /></figure>

CONTACT Simote T. Foliaki simote.foliaki@nih.gov

## ABSTRACT

**Introduction:** One of the fundamental problems of researching human prion diseases is access to live human brain tissue. Utilizing stem cell technology to produce human cerebral organoids (COs), which currently represent the nearest model to structured human brain tissue, offers a new avenue of investigation [1]. These organoids are self-organizing, structured regions analogous to human brain tissue that mature to contain multiple neuronal populations as well as astrocytes and oligodendrocytes [1, 2]. Importantly, these COs could be generated from donors who are carriers of mutations associated with familial prion diseases such as the E200K mutation. Herein we aimed to investigate the influence of E200K mutation that pre-dispose individuals to prion disease on neuronal electrical signalling.

**Methods:** COs have been generated from donors with E200K mutations (pre-disposed) and without (non-predisposed) mutations within the prion gene using the protocol described by Lancaster and Knoblick [1]. To measure the ability of these organoids to evoke spontaneous neuronal electrical signalling, they were sliced into 300 µm thick slices and mounted onto multi-electrode arrays (MEAs). MEAs are embedded with micro-electrodes to measure extracellular neuronal electrical spike (action potentials) and network communication throughout individual organoid. During the recording, the organoid slices were provided with adequate nutrients and oxygen through continuous superfusion with oxygenated artificial cerebrospinal fluid. To induce electrical signalling associated with cognition and behaviour, we exposed the slices to known stimulants and depressants of neuronal activity to determine their ability to express functions associated with memory formation and those related to memory loss [3, 4].

**Results and Conclusion:** We found that both non-predisposed and predisposed organoids exhibited active spontaneous neuronal function and network activity. Exposure of these organoids to glycine induced a persistently enhanced rate of neuronal spikes, a physiological correlate of memory formation called Long-Term Potentiation (LTP). Exposure of these organoids to glutamate/glycine induced LTP as well as Long-Term Depression (LTD), a physiological correlate of memory loss. Overall, we observed differences between the predisposed and non-predisposed organoids in neuronal excitability and response to depressants which

may influence excitotoxicity events and level of fatigue in neuronal activity in these organoids.

## References

[1] Lancaster, et al. Nat Protoc. 2014;9(10):2329-2340.

[2] Renner, et al. Embo J. 2017;36(10):1316-1329.

[3] Igartua, et al. Neuropharmacology. 2007;52(8):1586-1595.

[4] Hasegawa, et al. Sci Rep. 2015;5:7707.

## 82. The celecoxib derivative AR-12 induces autophagy and controls prion infection in vitro and in vivo

Basant A. Abdulrahmanª,ᵇ, Dalia Abdelazizª,ᶜ, Simrika Thapaª,ᶜ, Li Luª,ᶜ, Sabine Gilchᵇ,ᶜ and Hermann M. SchatzIª,ᶜ

CONATCT Basant A. Abdulrahman baabdulr@ucalgary.ca

## ABSTRACT

Prion diseases are fatal infectious neurodegenerative disorders that affect both humans and animals. The autocatalytic conversion of the cellular prion protein (PrPC) into the pathologic isoform PrPSc is a key feature in prion pathogenesis. AR-12 is an IND-approved derivative of celecoxib that demonstrated preclinical activity against several microbial diseases. Loss of neurons, astrogliosis and mild microglia activation are the main pathological features of prion diseases. This results in a progressive spongiform degeneration of the central nervous system (CNS), leading to ataxia, behavioural changes and, in humans, highly progressive loss of intellectual abilities. In the last two decades, great efforts have been made to establish treatment options for prion diseases. These included testing existing drugs for anti-prion activity in experimental models with only a few agents progressing to human studies of patients with prion diseases. Investigations to date have not resulted in a recognized/proven treatment for prion diseases. Recently, AR-12 (a.k.a. OSU-03012), a potent inhibitor of PDK-1 (phosphoinositide-dependent kinase 1) and autophagy stimulator, has been shown to facilitate clearance of misfolded proteins. The latter proposes AR-12 to be a

<footnote>ªNational Institute of Allergy and Infectious Diseases, NIH; ᵇCase Western Reserve University School of Medicine</footnote>

<footnote>ªDepartment of Comparative Biology & Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, Canada; ᵇDepartment of Ecosystem & Public Health, Faculty of Veterinary Medicine, University of Calgary, Calgary, Canada; ᵈCalgary Prion Research Unit, and Hotchkiss Brain Institute, University of Calgary, Calgary, Canada</footnote>
<figure><img src="image_52.png" /></figure>

potential therapeutic agent for neurodegenerative disorders. In our study, we investigated the role of AR-12 in controlling prion infection. We tested AR-12 in prion infected neuronal cells (ScN2a, ScCAD5) and non-neuronal cells (ScMEF). Immunoblotting and confocal microscopy results showed that AR-12 significantly reduced PrPSc levels after only 72 h of treatment. Furthermore, infected cells were cured of PrPSc after exposure of AR-12 for only 2 weeks. We partially attribute the influence of the AR-12 on prion propagation to autophagy stimulation. Furthermore, we tested the therapeutic effect of AR-12 in vivo in RML-infected mice. Interestingly, treatment with AR-12 (intraperitoneally and orally) significantly extended the survival of the treated animals compared to the control group, in line with our previous findings that drug-induced stimulation of autophagy has anti-prion effects in vitro. Taken together, this study demonstrates that AR-12 represents a potential new therapeutic agent for prion diseases and possibly protein misfolding disorders involving prion-like mechanisms.

**KEYWORDS:** Prions; PrPSc; Autophagy; AR-12

**83. Rationally designed, structure-based vaccine candidates targeting prion diseases**

Andrew Fangª, Xinli Tangª, Brian Tancownyª, Xiongyao Wangª* and Holger Willeª

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

CONTACT Andrew Fang jfang5@ualberta.ca

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

**ABSTRACT**

Prion diseases are caused by the misfolding of PrP<sup>C</sup> to PrP<sup>Sc</sup>, resulting in radical changes in secondary and tertiary structure. The lack of prophylactic or therapeutic vaccines means uncontrolled spread and an invariably fatal outcome for the host. No high-resolution structure for PrP<sup>Sc</sup> exists despite it having been studied extensively, but there is good evidence to suggest a four-rung β-solenoid structure. HET-s is a fungal prion protein that shares the β-solenoid fold with PrP<sup>Sc</sup>, albeit with only two-rungs. A four-rung β-solenoid version of HET-s, termed HET-2s, was engineered via a linker connecting the prion-forming domain of HET-s twice. Strategic placement of seven PrP-based

amino acids into HET-2s allowed us to mimic the surface structure of PrP<sup>Sc</sup> and marks a new, structure-based approach in prion vaccine design. Constructs were expressed in E. coli, purified, and assembled into amyloid fibrils morphologically indistinguishable from native HET-s fibrils. Vaccine candidates showing good fibrillization, and therefore adopting the proper β-solenoid fold, were used for immunization experiments. We recently showed that one of our vaccine candidates resulted in a specific immune response towards native PrP<sup>Sc</sup>.

Efficacy testing using an oral prion infection model in Syrian hamsters is ongoing to evaluate whether the vaccine candidate is effective against peripheral infection routes. Monoclonal antibodies (mAbs) that originated from the immunized mice were shown to recognize native PrP<sup>Sc</sup> in brain homogenates from all prion isolates that were tested. A competition ELISA that uses these mAbs is showing great potential as a diagnostic tool (See Tang *et al.* poster). Moreover, the ability of these mAbs to recognize native PrP<sup>Sc</sup> from various strains also reveals shared structural characteristics (See Wille *et al.* poster). Furthermore, the IgM and IgG clones are being sequenced and revealed unique complementarity-determining regions, indicating they all have different epitopes (See Rathod *et al.* poster). Lastly, this approach has been used to also design vaccines against other amyloid diseases, specifically targeting misfolded Aβ, α-synuclein and tau protein (See Flores-Fernandez *et al.* poster). Taken together, our novel vaccine design moves us one step closer to develop passive and active immunotherapies for the prion diseases.

**84. Stress granules "road to ruin" in neurodegenerative diseases**

Neelam Younas, Saima Zafar and Inga Zerr

Department of Neurology, Clinical Dementia Centre, University Medical Centre Goettingen(UMG), Germany

**ABSTRACT**

**Introduction:** Stress granules are membrane less cytoplasmic foci of mRNA and RNA binding proteins that are formed in the cell to cope with variety of stressors. Emerging evidences have shown disturbances in the dynamics of these non-membranous foci, as a pathophysiological element of amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and more
<figure><img src="image_56.png" /></figure>

potential therapeutic agent for neurodegenerative disorders. In our study, we investigated the role of AR-12 in controlling prion infection. We tested AR-12 in prion infected neuronal cells (ScN2a, ScCAD5) and non-neuronal cells (ScMEF). Immunoblotting and confocal microscopy results showed that AR-12 significantly reduced PrPSc levels after only 72 h of treatment. Furthermore, infected cells were cured of PrPSc after exposure of AR-12 for only 2 weeks. We partially attribute the influence of the AR-12 on prion propagation to autophagy stimulation. Furthermore, we tested the therapeutic effect of AR-12 in vivo in RML-infected mice. Interestingly, treatment with AR-12 (intraperitoneally and orally) significantly extended the survival of the treated animals compared to the control group, in line with our previous findings that drug-induced stimulation of autophagy has anti-prion effects in vitro. Taken together, this study demonstrates that AR-12 represents a potential new therapeutic agent for prion diseases and possibly protein misfolding disorders involving prion-like mechanisms.

**KEYWORDS:** Prions; PrPSc; Autophagy; AR-12

**83. Rationally designed, structure-based vaccine candidates targeting prion diseases**

Andrew Fangª, Xinli Tangª, Brian Tancownyª, Xiongyao Wangª* and Holger Willeª

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

CONTACT Andrew Fang jfang5@ualberta.ca

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

**ABSTRACT**

Prion diseases are caused by the misfolding of PrP<sup>C</sup> to PrP<sup>Sc</sup>, resulting in radical changes in secondary and tertiary structure. The lack of prophylactic or therapeutic vaccines means uncontrolled spread and an invariably fatal outcome for the host. No high-resolution structure for PrP<sup>Sc</sup> exists despite it having been studied extensively, but there is good evidence to suggest a four-rung β-solenoid structure. HET-s is a fungal prion protein that shares the β-solenoid fold with PrP<sup>Sc</sup>, albeit with only two-rungs. A four-rung β-solenoid version of HET-s, termed HET-2s, was engineered via a linker connecting the prion-forming domain of HET-s twice. Strategic placement of seven PrP-based

amino acids into HET-2s allowed us to mimic the surface structure of PrP<sup>Sc</sup> and marks a new, structure-based approach in prion vaccine design. Constructs were expressed in E. coli, purified, and assembled into amyloid fibrils morphologically indistinguishable from native HET-s fibrils. Vaccine candidates showing good fibrillization, and therefore adopting the proper β-solenoid fold, were used for immunization experiments. We recently showed that one of our vaccine candidates resulted in a specific immune response towards native PrP<sup>Sc</sup>.

Efficacy testing using an oral prion infection model in Syrian hamsters is ongoing to evaluate whether the vaccine candidate is effective against peripheral infection routes. Monoclonal antibodies (mAbs) that originated from the immunized mice were shown to recognize native PrP<sup>Sc</sup> in brain homogenates from all prion isolates that were tested. A competition ELISA that uses these mAbs is showing great potential as a diagnostic tool (See Tang *et al.* poster). Moreover, the ability of these mAbs to recognize native PrP<sup>Sc</sup> from various strains also reveals shared structural characteristics (See Wille *et al.* poster). Furthermore, the IgM and IgG clones are being sequenced and revealed unique complementarity-determining regions, indicating they all have different epitopes (See Rathod *et al.* poster). Lastly, this approach has been used to also design vaccines against other amyloid diseases, specifically targeting misfolded Aβ, α-synuclein and tau protein (See Flores-Fernandez *et al.* poster). Taken together, our novel vaccine design moves us one step closer to develop passive and active immunotherapies for the prion diseases.

**84. Stress granules "road to ruin" in neurodegenerative diseases**

Neelam Younas, Saima Zafar and Inga Zerr

Department of Neurology, Clinical Dementia Centre, University Medical Centre Goettingen(UMG), Germany

**ABSTRACT**

**Introduction:** Stress granules are membrane less cytoplasmic foci of mRNA and RNA binding proteins that are formed in the cell to cope with variety of stressors. Emerging evidences have shown disturbances in the dynamics of these non-membranous foci, as a pathophysiological element of amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and more
<figure><img src="image_53.png" /></figure>

recently in Alzheimer disease¹,². These evidences redirect research efforts towards understanding the role of stress granule biology in neurodegenerative diseases. In this study, we aim to explore the role of oxidative-stress to proteopathic disease linked proteins which are major hallmarks of neurodegenerative diseases.

**Methods:** For generation of Cell Stress Model, human cervical adenocarcinoma (HeLa) cell line was treated with 0.6mM sodium arsenite (a classical stress inducer) at 37°C for 60 min. Immunofluorescence was used for visualization of stress granules. Differential expression of selected target proteins was analysed in untreated (control) and treated cells (stressed) and in Human brain patients by Immunoblotting. Tau-Pathology Model was used to find out direct mechanistic link of target proteins to disease pathology.

**Results:** Interestingly, we found that aggregated prone proteins particularly phosphorylated form of Tau and Prion protein (Tau, PrP) were recruited to cytoplasmic stress granules after sodium arsenite treatment. Stress granules were identified by classical marker TIA-1 and PABP. To find out stress specific interactors of aggregated prone proteins interactome mapping of these proteins in untreated and stress induced cells revealed a common stress specific target, splicing factor proline, and glutamine rich (SFPQ) which is an important component of Neuronal transport granules. Here, we show that SFPQ is also a constituent of cytoplasmic stress granules. Arsenite treatment induced cytoplasmic SFPQ granules. Further to find out granule formation propensity of SFPQ, it was subjected to catGRANULE software which predicted a score of 1.66 showing a good propensity of SFPQ to form granules or liquid-liquid demixing. SFPQ was found to be significantly regulated in Alzheimer and CJD patient's brain in subtype dependent manner.

**Conclusions:** Our results highlight novel association/localization of aggregation prone proteins with stress granules linking cellular stress response to pathobiology of these proteins. Further differential regulation of SFPQ, its LLPS property and its localization in stress granules and interaction with Tau and Prion Protein links dots for SFPQ as might be crucible of aggregation via stress granules.

Supported by Helmholtz-Alberta Initiative – Neurodegenerative Diseases Research (HAI-NDR); Alberta innovates Bio solutions, and DZNE Goettingen.

## References

[1] Zhang P, et al. 2018;bioRxiv: 348,870.

[2] Vanderweyde, et al. Cell Reports. 2016;15:1455–1466.

## 85. Kinetics of prion seeding activity in brains of mice infected with mouse-adopted BSE prion

Yoshifumi Iwamaru, Yuichi Matsuura and Kohtaro Miyazawa

Prion Disease unit, Division of Transboundary Animal Disease, National Institute of Animal Health (NIAH), National Agriculture and Food Research Organization (NARO), Tsukuba, Japan

CONTACT gan@affrc.go.jp

## ABSTRACT

Prion diseases are fatal neurodegenerative disorders in humans and animals. The key event in the pathogenesis of these diseases is the conversion of host-encoded normal cellular prion protein (PrPC) into its pathogenic isoform (PrPSc) and its accumulation in the central nervous system. PrPSc is copurified with infectivity and thus thought to be the main constituent of the agent of prion diseases. PrPSc is partially resistant towards the proteinase K (PK) digestion and PK-resistant PrPSc (PrPres) has been used extensively as a surrogate marker for prion infection. However, several studies have revealed that prion infectivity is not always well-correlated with total PrPres levels. Real-time quaking-induced conversion (RT-QuIC) is one of newly developed in vitro amplification methods based on prion-seeded amyloid fibril formation by recombinant prion protein. According to a previous study, prion seeding activity appears to be correlated with prion infectivity than with total PrPres-levels. In our previous study of kinetics of mouse-adopted BSE prion propagation and PrPres accumulation, we demonstrated that the brain titer reached a plateau at 100 days post infection whereas PrPres continuously increased towards the end of the incubation period. In this study, we have proceeded to measure kinetics of prion seeding activity in the brain of these mice. We have used end-point dilution RT-QuIC on brain homogenates to produce a detailed measurement of seeding activity throughout the incubation period of the mice and evaluated the temporal relationships among seeding activity, infectivity, and PrPres levels.

## 86. Characterization of cellulose ether liposomes for the inhibition of prion formation in prion-infected cells

Keiko Nishizawaª, Kenta Teruyaª, Ayumi Ogumaª, Yuji Sakasegawaª, Hiroshi Kamitakaharaᵇ, Hermann Schatzlᶜ, Sabine Gilchᵈ and Katsumi Doh-uraª

<footnote>ªDepartment of Neurochemistry, Tohoku University Graduate School of Medicine, Sendai, Japan; ᵇDivision of Forest & Biomaterials Science Graduate School of Agriculture, Kyoto University, Kyoto, Japan; ᶜDepartment of Comparative Biology and Experimental Medicine,</footnote>
<figure><img src="image_57.png" /></figure>

recently in Alzheimer disease¹,². These evidences redirect research efforts towards understanding the role of stress granule biology in neurodegenerative diseases. In this study, we aim to explore the role of oxidative-stress to proteopathic disease linked proteins which are major hallmarks of neurodegenerative diseases.

**Methods:** For generation of Cell Stress Model, human cervical adenocarcinoma (HeLa) cell line was treated with 0.6mM sodium arsenite (a classical stress inducer) at 37°C for 60 min. Immunofluorescence was used for visualization of stress granules. Differential expression of selected target proteins was analysed in untreated (control) and treated cells (stressed) and in Human brain patients by Immunoblotting. Tau-Pathology Model was used to find out direct mechanistic link of target proteins to disease pathology.

**Results:** Interestingly, we found that aggregated prone proteins particularly phosphorylated form of Tau and Prion protein (Tau, PrP) were recruited to cytoplasmic stress granules after sodium arsenite treatment. Stress granules were identified by classical marker TIA-1 and PABP. To find out stress specific interactors of aggregated prone proteins interactome mapping of these proteins in untreated and stress induced cells revealed a common stress specific target, splicing factor proline, and glutamine rich (SFPQ) which is an important component of Neuronal transport granules. Here, we show that SFPQ is also a constituent of cytoplasmic stress granules. Arsenite treatment induced cytoplasmic SFPQ granules. Further to find out granule formation propensity of SFPQ, it was subjected to catGRANULE software which predicted a score of 1.66 showing a good propensity of SFPQ to form granules or liquid-liquid demixing. SFPQ was found to be significantly regulated in Alzheimer and CJD patient's brain in subtype dependent manner.

**Conclusions:** Our results highlight novel association/localization of aggregation prone proteins with stress granules linking cellular stress response to pathobiology of these proteins. Further differential regulation of SFPQ, its LLPS property and its localization in stress granules and interaction with Tau and Prion Protein links dots for SFPQ as might be crucible of aggregation via stress granules.

Supported by Helmholtz-Alberta Initiative – Neurodegenerative Diseases Research (HAI-NDR); Alberta innovates Bio solutions, and DZNE Goettingen.

## References

[1] Zhang P, et al. 2018;bioRxiv: 348,870.

[2] Vanderweyde, et al. Cell Reports. 2016;15:1455-1466.

## 85. Kinetics of prion seeding activity in brains of mice infected with mouse-adopted BSE prion

Yoshifumi Iwamaru, Yuichi Matsuura and Kohtaro Miyazawa

Prion Disease unit, Division of Transboundary Animal Disease, National Institute of Animal Health (NIAH), National Agriculture and Food Research Organization (NARO), Tsukuba, Japan

CONTACT gan@affrc.go.jp

## ABSTRACT

Prion diseases are fatal neurodegenerative disorders in humans and animals. The key event in the pathogenesis of these diseases is the conversion of host-encoded normal cellular prion protein (PrPC) into its pathogenic isoform (PrPSc) and its accumulation in the central nervous system. PrPSc is copurified with infectivity and thus thought to be the main constituent of the agent of prion diseases. PrPSc is partially resistant towards the proteinase K (PK) digestion and PK-resistant PrPSc (PrPres) has been used extensively as a surrogate marker for prion infection. However, several studies have revealed that prion infectivity is not always well-correlated with total PrPres levels. Real-time quaking-induced conversion (RT-QuIC) is one of newly developed in vitro amplification methods based on prion-seeded amyloid fibril formation by recombinant prion protein. According to a previous study, prion seeding activity appears to be correlated with prion infectivity than with total PrPres-levels. In our previous study of kinetics of mouse-adopted BSE prion propagation and PrPres accumulation, we demonstrated that the brain titer reached a plateau at 100 days post infection whereas PrPres continuously increased towards the end of the incubation period. In this study, we have proceeded to measure kinetics of prion seeding activity in the brain of these mice. We have used end-point dilution RT-QuIC on brain homogenates to produce a detailed measurement of seeding activity throughout the incubation period of the mice and evaluated the temporal relationships among seeding activity, infectivity, and PrPres levels.

## 86. Characterization of cellulose ether liposomes for the inhibition of prion formation in prion-infected cells

Keiko Nishizawaª, Kenta Teruyaª, Ayumi Ogumaª, Yuji Sakasegawaª, Hiroshi Kamitakaharaᵇ, Hermann Schatzlᶜ, Sabine Gilchᵈ and Katsumi Doh-uraª

<footnote>ªDepartment of Neurochemistry, Tohoku University Graduate School of Medicine, Sendai, Japan; ᵇDivision of Forest & Biomaterials Science Graduate School of Agriculture, Kyoto University, Kyoto, Japan; ᶜDepartment of Comparative Biology and Experimental Medicine,</footnote>
<figure><img src="image_54.png" /></figure>

Faculty of Veterinary Medicine; Hotchkiss Brain Institute; University of Calgary, Canada; ªDepartment of Ecosystem and Public Health, Calgary Prion Research Unit, Faculty of Veterinary Medicine, Hotchkiss Brain Institute, University of Calgary, Calgary, Canada

CONTACT Katsumi Doh-ura doh-ura@med.tohoku.ac.jp

## ABSTRACT

Our previous study demonstrated that a single peripheral administration of cellulose ethers (CE) dose-dependently prolonged the survival of prion-infected rodents when immediately administered after prion infection or even 1 year prior to infection. Additionally, CE inhibited prion formation of persistently prion-infected cells in a manner parallel to the *in vivo* efficacy. However, the mechanism of anti-prion action of CE is not fully understood. Even in the most effective CE, considerable doses, such as few mg/gm body weight in rodents or tens of mg/ml in cell culture media are needed for obtaining substantial effects. Therefore, improvement of the bioavailability is required for the practical use of CE. In this study, we prepared CE-loaded liposomes and examined the anti-prion activity in prion-infected cells to compare the effect of CE-loaded liposomes with that of unformulated CE on anti-prion activity. We also modified CE-loaded liposomes and investigated the uptake levels in prion-infected and macrophage-like cells, and then compared the uptake levels with anti-prion activity levels to help elucidate the anti-prion mechanism of CE-loaded liposomes. The liposomal formulation reduced the EC₅₀ dose of CE by <1/200-fold in prion-infected cells. Compared to empty liposomes, CE-loaded liposomes were taken up much more highly by prion-infected cells and less by macrophage-like cells. Phosphatidylserine modification reduced the uptake of CE-loaded liposomes in prion-infected cells and did not change the anti-prion activity, whereas increased the uptake in macrophage-like cells. Polyethylene glycol modification reduced the uptake of CE-loaded liposomes in both types of cells and reduced the anti-prion activity in prion-infected cells. These results suggest that a liposomal formulation of CE is more practical than unformulated CE and showed that the CE-loaded liposome uptake levels in prion-infected cells were not associated with anti-prion activity. Although further improvement of the stealth function against phagocytic cells is needed, the liposomal formulation is useful to improve CE efficacy and elucidate the mechanism of CE action.

## 87. Nascent β structure in the elongated hydrophobic region of a Gerstmann-Sträussler-Scheinker PrP Allele

Ze-Lin Fu<sup>a,b</sup>, Peter C. Holmes<sup>b</sup>, David Westaway<sup>a,b</sup> and Brian D. Sykes<sup>b</sup>

<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

## ABSTRACT

**Objective:** To understand the molecular basis of the misfolding of a novel prion protein (PrP) mutant that causes Gerstmann-Straussler-Scheinker (GSS) disease. This mutant designated 'HRdup' was defined in a 34-year-old GSS patient and contains an 8-amino-acid duplication (LGGLGGYV) after Val129. This allele encompasses both part of the glycine-rich portion of the hydrophobic region (HR) and all the first beta strand and is thus distinct from other GSS mutations that comprise missense mutations or insertions of extra octarepeats. Transgenic (Tg) mice expressing HRdup PrP develop a spontaneous neurological syndrome and proteinase K digestion of brain homogenates from these mice shows a pathognomonic GSS band at ~8kDa [1].

**Methods:** For structural analysis, we purified <sup>13</sup>C, <sup>15</sup>N double-labelled recombinant mPrP HRdup (118-231) along with two control alleles, mPrP V128 (118-231) and mPrP WT (118-231). Using 3D NMR experiments performed at pH 5.0 and in the absence of added co-factors, we assigned the NMR spectra of both HRdup and V128; therefore, determined the secondary structure of these two proteins and explored the molecular motions and dynamics of HRdup and V128. The secondary structure prediction programmes and MD simulation were also employed to predict and verify the conformation of the N-terminus of HRdup.

**Results:** The NMR experiments showed that HRdup has the same canonical globular PrP<sup>C</sup> structure as V128, and that the elongated N-terminus of HRdup displayed significant β-propensity while maintaining its high flexibility. MD simulation depicted this nascent β-structure in the hydrophobic region as a β-turn. In addition, we observed that a methionine/valine polymorphism at codon 128 (equivalent to codon 129 in humans) in the presence of oligomerization facilitated by high protein concentrations affected conformational exchange dynamics at residue G130 [2].

**Conclusions:** We conclude that the novel β-structure at the elongated hydrophobic region of HRdup is the root cause of spontaneous misfolding of HRdup PrP. As the exchange dynamics of G130 also suggests the potential
<figure><img src="image_58.png" /></figure>

Faculty of Veterinary Medicine; Hotchkiss Brain Institute; University of Calgary, Canada; ªDepartment of Ecosystem and Public Health, Calgary Prion Research Unit, Faculty of Veterinary Medicine, Hotchkiss Brain Institute, University of Calgary, Calgary, Canada

CONTACT Katsumi Doh-ura doh-ura@med.tohoku.ac.jp

## ABSTRACT

Our previous study demonstrated that a single peripheral administration of cellulose ethers (CE) dose-dependently prolonged the survival of prion-infected rodents when immediately administered after prion infection or even 1 year prior to infection. Additionally, CE inhibited prion formation of persistently prion-infected cells in a manner parallel to the *in vivo* efficacy. However, the mechanism of anti-prion action of CE is not fully understood. Even in the most effective CE, considerable doses, such as few mg/gm body weight in rodents or tens of mg/ml in cell culture media are needed for obtaining substantial effects. Therefore, improvement of the bioavailability is required for the practical use of CE. In this study, we prepared CE-loaded liposomes and examined the anti-prion activity in prion-infected cells to compare the effect of CE-loaded liposomes with that of unformulated CE on anti-prion activity. We also modified CE-loaded liposomes and investigated the uptake levels in prion-infected and macrophage-like cells, and then compared the uptake levels with anti-prion activity levels to help elucidate the anti-prion mechanism of CE-loaded liposomes. The liposomal formulation reduced the EC₅₀ dose of CE by <1/200-fold in prion-infected cells. Compared to empty liposomes, CE-loaded liposomes were taken up much more highly by prion-infected cells and less by macrophage-like cells. Phosphatidylserine modification reduced the uptake of CE-loaded liposomes in prion-infected cells and did not change the anti-prion activity, whereas increased the uptake in macrophage-like cells. Polyethylene glycol modification reduced the uptake of CE-loaded liposomes in both types of cells and reduced the anti-prion activity in prion-infected cells. These results suggest that a liposomal formulation of CE is more practical than unformulated CE and showed that the CE-loaded liposome uptake levels in prion-infected cells were not associated with anti-prion activity. Although further improvement of the stealth function against phagocytic cells is needed, the liposomal formulation is useful to improve CE efficacy and elucidate the mechanism of CE action.

## 87. Nascent β structure in the elongated hydrophobic region of a Gerstmann-Sträussler-Scheinker PrP Allele

Ze-Lin Fu<sup>a,b</sup>, Peter C. Holmes<sup>b</sup>, David Westaway<sup>a,b</sup> and Brian D. Sykes<sup>b</sup>

<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

## ABSTRACT

**Objective:** To understand the molecular basis of the misfolding of a novel prion protein (PrP) mutant that causes Gerstmann-Straussler-Scheinker (GSS) disease. This mutant designated 'HRdup' was defined in a 34-year-old GSS patient and contains an 8-amino-acid duplication (LGGLGGYV) after Val129. This allele encompasses both part of the glycine-rich portion of the hydrophobic region (HR) and all the first beta strand and is thus distinct from other GSS mutations that comprise missense mutations or insertions of extra octarepeats. Transgenic (Tg) mice expressing HRdup PrP develop a spontaneous neurological syndrome and proteinase K digestion of brain homogenates from these mice shows a pathognomonic GSS band at ~8kDa [1].

**Methods:** For structural analysis, we purified <sup>13</sup>C, <sup>15</sup>N double-labelled recombinant mPrP HRdup (118-231) along with two control alleles, mPrP V128 (118-231) and mPrP WT (118-231). Using 3D NMR experiments performed at pH 5.0 and in the absence of added co-factors, we assigned the NMR spectra of both HRdup and V128; therefore, determined the secondary structure of these two proteins and explored the molecular motions and dynamics of HRdup and V128. The secondary structure prediction programmes and MD simulation were also employed to predict and verify the conformation of the N-terminus of HRdup.

**Results:** The NMR experiments showed that HRdup has the same canonical globular PrP<sup>C</sup> structure as V128, and that the elongated N-terminus of HRdup displayed significant β-propensity while maintaining its high flexibility. MD simulation depicted this nascent β-structure in the hydrophobic region as a β-turn. In addition, we observed that a methionine/valine polymorphism at codon 128 (equivalent to codon 129 in humans) in the presence of oligomerization facilitated by high protein concentrations affected conformational exchange dynamics at residue G130 [2].

**Conclusions:** We conclude that the novel β-structure at the elongated hydrophobic region of HRdup is the root cause of spontaneous misfolding of HRdup PrP. As the exchange dynamics of G130 also suggests the potential
<figure><img src="image_55.png" /></figure>

involvement of the canonical β-sheet in the initializa-
tion of PrP misfolding, we hypothesize that the hydro-
phobic region of HRdup can adopt a fully extended
configuration, fold back and develop into a three-
strand antiparallel β-structure that incorporates the
canonical β-sheet. This proposed structure is highly
consistent with the structure observed in full length
hPrP when crystalized with nano-body at its N-term-
inal end [3].

References

[1] Mercer RCC, et al. PLOS Pathogen. 2018;14(1):e1006826.

[2] Fu Z-F, et al. Submitted for publication.

[3] Abskharon RNN, et al. JACS 2014;136(2):937-944.

**88. Sporadic CJD in pregnancy: an interdisciplinary success story**

Collin Lukª, Allison Thieleᵇ, Christina Orru-Grovemanᶜ, Byron Caugheyᶜ and Valerie L. Simª,ᵈ

ªDepartment of Medicine, Division of Neurology, Faculty of Medicine & Dentistry, University of Alberta; ᵇDepartment of Obstetrics and Gynaecology, Division of Maternal Fetal Medicine, Faculty of Medicine & Dentistry, University of Alberta; ᶜRocky Mountain Laboratories, NIAID, National Institutes of Health, Hamilton, MT, USA; ᵈCentre for Prions and Protein Folding Diseases, University of Alberta

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

ABSTRACT

Sporadic Creutzfeldt-Jakob disease (sCJD) affects 1–2 people per million per year, with a peak incidence in the 7th decade of life. Rarely, CJD can affect women of childbearing age.

There are seven previously reported cases of CJD (six sporadic, one iatrogenic) in pregnancy with five healthy deliveries and no evidence of vertical transmission to date.1-5 In these reports, immunohistochemical screens of placental tissue were negative for PrPSc, but in the one study that tested for placental infectivity, prion disease was transmitted to five of eight cerebrally inoculated Balb mice.2 The possibility that placenta may contain infectious prions has implications for vertical transmission and risk management of delivery. We present a 35 year old G2P1 woman with probable sCJD who presented in her 10th gestational week with a 7-month history of progressive memory impairment and visuospatial difficulties. Initial investigations, including CSF studies, were negative for autoimmune, inflammatory, infectious, and neoplastic conditions. A positive ds-DNA raised the possibility of

lupus, for which she was started on Plaquenil, but no
other clinical criteria were identified. An electroencepha-
logram demonstrated bilateral diffuse slowing. Brain MRI
was delayed until the beginning of the 2nd trimester out
of theoretical concern for foetal exposure. The MRI
showed restricted diffusion of basal ganglia and cortical
ribboning consistent with Creutzfeldt-Jakob disease. A
repeat CSF revealed elevated 14-3-3 (>80 000 AU/mL),
elevated Tau protein (>10 513 pg/mL) and positive EP-
QuIC assay. There was no PRNP gene mutation. The rate
of clinical progression required extensive interdisciplinary
management to preserve the pregnancy and safely plan
for a number of possible obstetric scenarios. A Kaofeed
was required to maintain nutrition. Oxygen was provided
by nasal prongs; intubation and ventilation was not
required. Fevers and tachycardia developed but without
evidence of infection, suggesting central autonomic dys-
regulation. Ultimately, a healthy baby was delivered via C-
section at 36 weeks 3 days gestation, with no obstetric or
post-anaesthetic complications. Cord blood, amniotic
fluid and placental tissue were collected for evaluation
by RT-QuIC. The patient passed away 24 days after deliv-
ery. A discussion of diagnostic challenges, management
strategies and QuIC results will be presented.

**References**

[1] Xiao X, et al. Am J Pathol. 2009;174(5):1602-1608

[2] Tamai Y, et al. N Engl J Med. 1992;327(9):649.

[3] Sperling R, et al. J Obstet Gynecol Neonatal Nurs. 2005;34(5):546-550.

[4] Di Gangi S, et al. J Matern Fetal Neonatal Med. 2015;28 (3):254-261.

[5] Dalmas AF, et al. Ann Fr Anesth Reanim. 2010;29 (11):815-817.

89. Exploiting their sweet tooth: sugars against prions

M. Kumarª,ᵇ, Garrett J.B. Molnerᶜ and Valerie L. Simª,ᵇ,

ªDepartment of Medicine, Division of Neurology, Faculty of Medicine & Dentistry, University of Alberta; ᵇCentre for Prions and Protein Folding Diseases, University of Alberta; ᶜDepartment of Medical Microbiology, Faculty of Medicine & Dentistry, University of Alberta

CONTACT M. Kumar manjeet2@ualberta.ca

ABSTRACT

Endoplasmic reticulum-associated degradation (ERAD) is critical to cellular protein homoestasis. The accumulation of unfolded or misfolded proteins in the ER challenges this proteostasis and has been
<figure><img src="image_59.png" /></figure>

involvement of the canonical β-sheet in the initializa-
tion of PrP misfolding, we hypothesize that the hydro-
phobic region of HRdup can adopt a fully extended
configuration, fold back and develop into a three-
strand antiparallel β-structure that incorporates the
canonical β-sheet. This proposed structure is highly
consistent with the structure observed in full length
hPrP when crystalized with nano-body at its N-term-
inal end [3].

References

[1] Mercer RCC, et al. PLOS Pathogen. 2018;14(1):e1006826.

[2] Fu Z-F, et al. Submitted for publication.

[3] Abskharon RNN, et al. JACS 2014;136(2):937-944.

88. *Sporadic CJD in pregnancy: an interdisciplinary success story*

Collin Lukª, Allison Thieleᵇ, Christina Orru-Grovemanᶜ, Byron Caugheyᶜ and Valerie L. Simª,ᵈ

ªDepartment of Medicine, Division of Neurology, Faculty of Medicine & Dentistry, University of Alberta; ᵇDepartment of Obstetrics and Gynaecology, Division of Maternal Fetal Medicine, Faculty of Medicine & Dentistry, University of Alberta; ᶜRocky Mountain Laboratories, NIAID, National Institutes of Health, Hamilton, MT, USA; ᵈCentre for Prions and Protein Folding Diseases, University of Alberta

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

ABSTRACT

Sporadic Creutzfeldt-Jakob disease (sCJD) affects 1-2 people per million per year, with a peak incidence in the 7th decade of life. Rarely, CJD can affect women of childbearing age.

There are seven previously reported cases of CJD (six sporadic, one iatrogenic) in pregnancy with five healthy deliveries and no evidence of vertical transmission to date.1-5 In these reports, immunohistochemical screens of placental tissue were negative for PrPSc, but in the one study that tested for placental infectivity, prion disease was transmitted to five of eight cerebrally inoculated Balb mice.2 The possibility that placenta may contain infectious prions has implications for vertical transmission and risk management of delivery. We present a 35 year old G2P1 woman with probable sCJD who presented in her 10th gestational week with a 7-month history of progressive memory impairment and visuospatial difficulties. Initial investigations, including CSF studies, were negative for autoimmune, inflammatory, infectious, and neoplastic conditions. A positive ds-DNA raised the possibility of

lupus, for which she was started on Plaquenil, but no
other clinical criteria were identified. An electroencepha-
logram demonstrated bilateral diffuse slowing. Brain MRI
was delayed until the beginning of the 2nd trimester out
of theoretical concern for foetal exposure. The MRI
showed restricted diffusion of basal ganglia and cortical
ribboning consistent with Creutzfeldt-Jakob disease. A
repeat CSF revealed elevated 14-3-3 (>80 000 AU/mL),
elevated Tau protein (>10 513 pg/mL) and positive EP-
QuIC assay. There was no PRNP gene mutation. The rate
of clinical progression required extensive interdisciplinary
management to preserve the pregnancy and safely plan
for a number of possible obstetric scenarios. A Kaofeed
was required to maintain nutrition. Oxygen was provided
by nasal prongs; intubation and ventilation was not
required. Fevers and tachycardia developed but without
evidence of infection, suggesting central autonomic dys-
regulation. Ultimately, a healthy baby was delivered via C-
section at 36 weeks 3 days gestation, with no obstetric or
post-anaesthetic complications. Cord blood, amniotic
fluid and placental tissue were collected for evaluation
by RT-QuIC. The patient passed away 24 days after deliv-
ery. A discussion of diagnostic challenges, management
strategies and QuIC results will be presented.

References

[1] Xiao X, et al. Am J Pathol. 2009;174(5):1602-1608

[2] Tamai Y, et al. N Engl J Med. 1992;327(9):649.

[3] Sperling R, et al. J Obstet Gynecol Neonatal Nurs. 2005;34(5):546-550.

[4] Di Gangi S, et al. J Matern Fetal Neonatal Med. 2015;28 (3):254-261.

[5] Dalmas AF, et al. Ann Fr Anesth Reanim. 2010;29 (11):815-817.

89. Exploiting their sweet tooth: sugars against prions

M. Kumarª,ᵇ, Garrett J.B. Molnerᶜ and Valerie L. Simª,ᵇ,

ªDepartment of Medicine, Division of Neurology, Faculty of Medicine & Dentistry, University of Alberta; ᵇCentre for Prions and Protein Folding Diseases, University of Alberta; ᶜDepartment of Medical Microbiology, Faculty of Medicine & Dentistry, University of Alberta

CONTACT M. Kumar manjeet2@ualberta.ca

ABSTRACT

Endoplasmic reticulum-associated degradation (ERAD) is critical to cellular protein homoestasis. The accumulation of unfolded or misfolded proteins in the ER challenges this proteostasis and has been
<figure><img src="image_56.png" /></figure>

implicated in neurodegeneration1. In prion disease, cellular prion protein (PrPC) is converted into a β-sheet rich infectious form (PrPSc)2. PrPC is a membranous protein that is variably N-glycosylated and, like other glycoproteins, depends on enzymes such as glucosidase I, II and glucosyltransferase for maturation of sugar residues as well as proper folding of the protein and its ultimate localization. Failure to properly glycosylate a membrane protein within the ER induces proteostasis stress in the ER and activates ER quality control machinery1,3,4. We hypothesize that trimming the glycans during PrPC synthesis will increase PrP levels in the ER and activate ER-associated degradation (ERAD). In the context of prion infection, this could both reduce available substrate for conversion and enhance clearance of misfolded PrP. We inhibited α- and β-glucosidases with four sugar analogues in CAD5 cells chronically infected with RML strain prions. Moderate dosages of individual analogues resulted in complete eradication of PrPSc within 72 h. Interestingly, treating with lower dosages in combination generated different PK-resistant prion species. Immunoblotting experiments also indicated a change in the protein level of OS-9, a major player in ER quality control and ERAD. OS-9 binds terminally misfolded non-glycoproteins as well as improperly folded glycoproteins and transfers them to ubiquitination machinery for targeted degradation5-7. We speculate that truncation of N-linked glycans on PrP with the combination of lower dose sugar analogues resulted in the formation of modified improperly folded mono- and di-glycosylated PrPC with altered conformational repertoire. These types of sugar analogues have high oral bioavailability, can easily penetrate the central nervous system, have extensively characterized biological and pharmacokinetic properties. As such, they may have therapeutic utility for prion diseases at moderate dose. In addition, they represent a novel tool for the experimental manipulation of prion strains.

## References

[1] Rendleman, et al. eLife. 2018;7:e39054

[2] Prusiner SB. Proc Natl Acad Sci. 1998;95:13,363–13,383

[3] Fagioli, et al. J Biol Chem. 2001;276:12,885–12,892;4.

[4] Smith, et al. Science. 2011;334:1086–1090.

[5] Bernasconi, et al. J Biol Chem. 2008;283:16,446–16,454;6.

[6] Hosokawa, et al. J Biol Chem. 2009;284:17,061–17068.

[7] Alcock, et al. J Mol Biol. 2009;385:1032–1042.

# 90. Estimation of the binding sites of inhibitory factors on recombinant PrP molecule by RT-QuIC

Akio Suzuki, Kazuhei Sawada, Takeshi Yamasaki and Motohiro Horiuchi

Laboratory of Veterinary Hygiene, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan

CONTACT Akio Suzukisusan@vetmed.hokudai.ac.jp

## ABSTRACT

**Background:** Real-time quaking-induced conversion (RT-QuIC) is one of several highly sensitive methods for the detection of prions. However, the reaction is easily interfered by inhibitory factors in tissue homogenates. We previously showed that recombinant cervid PrP (rCerPrP) can react with prions even in the presence of tissue homogenates (Prion 2017, P152). To clarify the region of the PrP molecule that is affected by inhibitory factors, we produced a variety of rPrP mutants and analysed the reactivity of the PrP mutants to H- and L-BSE prions by RT-QuIC.

**Material and Methods:** Full-length recombinant sheep (ARQ) PrP (rShPrP), mouse PrP (rMoPrP), rCerPrP, and their C-terminal chimeras (rCerPrP-MoCterm and rMoPrP-CerCterm), and their mutants that have point-mutations at ShPrP-specific amino acid (aa) 98 S and/or CerPrP-specific 173 N and 177 T (rMoPrP-S169NCer/N173TCer, rMoPrP-T94SSh/N96SSh/S169NCer/N173TCer and rCerPrP-N173SMo/T177NMo/, rCerPrP-T98SSh/N173SMo/T177NMo) were used as substrates. Brain homogenates (BH) of cattle infected with H-BSE and L-BSE were serially diluted with PBS or normal cattle BH and used as seeds. RT-QuIC reactions were performed under conditions containing 10 mM Na-phosphate (pH 7.4), 500 mM NaCl and 0.001% SDS at 37 °C.

**Results:** rShPrP, rMoPrP and rCerPrP could comparably detect H-BSE and L-BSE diluted at $10^{-4}$–$10^{-7}$ with PBS. However, the reaction of rShPrP with L-BSE was delayed for 13.4 h on average and those of rMoPrP with H-BSE and L-BSE were delayed for 27.4 h and completely inhibited by 0.1% normal BH, respectively. Compared to authentic rPrPs, the reaction of rCerPrP-MoCterm with L-BSE in the presence of normal BH was delayed for 10.7 h, whereas that of rMoPrP-CerCterm was shortened by 21.9 h. These results suggest that the C-terminus of rMoPrP is more affected by the inhibitory factors compared with those of rCerPrP. On the other hand, the reaction of rCerPrP-N173SMo/T177NMo/and rCerPrP-T98SSh/N173SMo/T177NMo with L-BSE was delayed for 10.8 h and 13.8 h compared to rCerPrP, whereas those of rMoPrP-S169NCer/N173TCer and rMoPrP-T94SSh/
<figure><img src="image_60.png" /></figure>

implicated in neurodegeneration1. In prion disease, cellular prion protein (PrPC) is converted into a β-sheet rich infectious form (PrPSc)2. PrPC is a membranous protein that is variably N-glycosylated and, like other glycoproteins, depends on enzymes such as glucosidase I, II and glucosyltransferase for maturation of sugar residues as well as proper folding of the protein and its ultimate localization. Failure to properly glycosylate a membrane protein within the ER induces proteostasis stress in the ER and activates ER quality control machinery1,3,4. We hypothesize that trimming the glycans during PrPC synthesis will increase PrP levels in the ER and activate ER-associated degradation (ERAD). In the context of prion infection, this could both reduce available substrate for conversion and enhance clearance of misfolded PrP. We inhibited α- and β-glucosidases with four sugar analogues in CAD5 cells chronically infected with RML strain prions. Moderate dosages of individual analogues resulted in complete eradication of PrPSc within 72 h. Interestingly, treating with lower dosages in combination generated different PK-resistant prion species. Immunoblotting experiments also indicated a change in the protein level of OS-9, a major player in ER quality control and ERAD. OS-9 binds terminally misfolded non-glycoproteins as well as improperly folded glycoproteins and transfers them to ubiquitination machinery for targeted degradation5-7. We speculate that truncation of N-linked glycans on PrP with the combination of lower dose sugar analogues resulted in the formation of modified improperly folded mono- and di-glycosylated PrPC with altered conformational repertoire. These types of sugar analogues have high oral bioavailability, can easily penetrate the central nervous system, have extensively characterized biological and pharmacokinetic properties. As such, they may have therapeutic utility for prion diseases at moderate dose. In addition, they represent a novel tool for the experimental manipulation of prion strains.

## References

[1] Rendleman, et al. eLife. 2018;7:e39054

[2] Prusiner SB. Proc Natl Acad Sci. 1998;95:13,363–13,383

[3] Fagioli, et al. J Biol Chem. 2001;276:12,885–12,892;4.

[4] Smith, et al. Science. 2011;334:1086–1090.

[5] Bernasconi, et al. J Biol Chem. 2008;283:16,446–16,454;6.

[6] Hosokawa, et al. J Biol Chem. 2009;284:17,061–17068.

[7] Alcock, et al. J Mol Biol. 2009;385:1032–1042.

# 90. Estimation of the binding sites of inhibitory factors on recombinant PrP molecule by RT-QuIC

Akio Suzuki, Kazuhei Sawada, Takeshi Yamasaki and Motohiro Horiuchi

Laboratory of Veterinary Hygiene, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan

CONTACT Akio Suzukisusan@vetmed.hokudai.ac.jp

## ABSTRACT

**Background:** Real-time quaking-induced conversion (RT-QuIC) is one of several highly sensitive methods for the detection of prions. However, the reaction is easily interfered by inhibitory factors in tissue homogenates. We previously showed that recombinant cervid PrP (rCerPrP) can react with prions even in the presence of tissue homogenates (Prion 2017, P152). To clarify the region of the PrP molecule that is affected by inhibitory factors, we produced a variety of rPrP mutants and analysed the reactivity of the PrP mutants to H- and L-BSE prions by RT-QuIC.

**Material and Methods:** Full-length recombinant sheep (ARQ) PrP (rShPrP), mouse PrP (rMoPrP), rCerPrP, and their C-terminal chimeras (rCerPrP-MoCterm and rMoPrP-CerCterm), and their mutants that have point-mutations at ShPrP-specific amino acid (aa) 98 S and/or CerPrP-specific 173 N and 177 T (rMoPrP-S169NCer/N173TCer, rMoPrP-T94SSh/N96SSh/S169NCer/N173TCer and rCerPrP-N173SMo/T177NMo/, rCerPrP-T98SSh/N173SMo/T177NMo) were used as substrates. Brain homogenates (BH) of cattle infected with H-BSE and L-BSE were serially diluted with PBS or normal cattle BH and used as seeds. RT-QuIC reactions were performed under conditions containing 10 mM Na-phosphate (pH 7.4), 500 mM NaCl and 0.001% SDS at 37 °C.

**Results:** rShPrP, rMoPrP and rCerPrP could comparably detect H-BSE and L-BSE diluted at $10^{-4}$–$10^{-7}$ with PBS. However, the reaction of rShPrP with L-BSE was delayed for 13.4 h on average and those of rMoPrP with H-BSE and L-BSE were delayed for 27.4 h and completely inhibited by 0.1% normal BH, respectively. Compared to authentic rPrPs, the reaction of rCerPrP-MoCterm with L-BSE in the presence of normal BH was delayed for 10.7 h, whereas that of rMoPrP-CerCterm was shortened by 21.9 h. These results suggest that the C-terminus of rMoPrP is more affected by the inhibitory factors compared with those of rCerPrP. On the other hand, the reaction of rCerPrP-N173SMo/T177NMo/and rCerPrP-T98SSh/N173SMo/T177NMo with L-BSE was delayed for 10.8 h and 13.8 h compared to rCerPrP, whereas those of rMoPrP-S169NCer/N173TCer and rMoPrP-T94SSh/
<figure><img src="image_57.png" /></figure>

N96S<sub>Sh</sub>/S169N<sub>Cer</sub>/N173T<sub>Cer</sub> was shortened by 11.5 h and 14.0 h compared to rMoPrP. Taken together, the inhibitory factors may also interfere with the region including CerPrP specific N173, and T177.

**Conclusion:** Our results suggest that conversion efficiency in the RT-QuIC reaction may be interfered with by the inhibitory factors at least at two regions of the PrP molecule, one is the C-terminus and the other is the region including CerPrP-specific aa 173 N and 177 T. These regions have been shown to be adjacent in the three-dimensional structure of PrP<sup>C</sup>; therefore, inhibitory factors may bind to the site including these regions.

**91. PrP regulates neural stem cell senescence through epidermal growth factor receptor**

Bradley R. Groveman, Simote T. Foliaki, Aleksandar R. Wood, Brent Race and Cathryn L. Haigh
Rocky Mountain Laboratories, NIAID, NIH, Hamilton, MT, USA

CONTACT Bradley R. Groveman Bradley.groveman@nih.gov

**ABSTRACT**

The prion protein (PrP) is expressed in a wide variety of progenitor cells and is known to be involved in neural stem cell self-renewal processes. The mechanisms by which PrP acts to modulate cell homeostasis have, however, not been fully elucidated. Recent studies have shown that PrP has a direct role in preventing premature senescence [1], and that knockdown of PrP increases senescence markers and decreases the cell proliferative potential. PrP has also been shown to regulate epidermal growth factor receptor (EGFR) through Notch signalling. Notch signalling, which can upregulate EGFR, is disrupted in the absence of PrP [2], leading to a reduction in EGFR expression. Furthermore, inhibition of EGFR or depletion of EGF have been shown to suppress senescence in proliferative cells [3]. Herein we attempt to show the role of PrP in regulating cellular senescence through the EGFR pathway.

**KEYWORDS:** Prion; PrP; neural stem cell; senescence; epidermal growth factor receptor; EGFR

**References**

[1] Boilan E, et al. Mech Ageing Dev. 2018;170:106-113.

[2] Martin-Lannerée S, et al. Stem Cells. 2017;35(3):754-765.

[3] Alexander PB, et al. Cell Res. 2015;25(1):135-138.

**92. Region-specific interference of PrP<sup>Sc</sup> accumulation in spleen and brain by poly-L-arginine (PLR)**

Jieun Kim<sup>a</sup>, Taeyeon Kim<sup>b</sup>, A-ran Kim<sup>a</sup>, Hakmin Lee<sup>a</sup>, Trang H. T. Trinh<sup>a</sup>, Junwu Shin<sup>a</sup>, Sungeun Lee<sup>a</sup> and Chongsuk Ryou<sup>a</sup>

<sup>a</sup>Department of Pharmacy, College of Pharmacy and Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan, Gyeonggi-do, Republic of Korea; <sup>b</sup>Division of Developmental Biology and Physiology, School of Biosciences and Chemistry, Institute for Basic science, Sungshin University, Seoul, Republic of Korea.

CONTACT Chongsuk Ryou cryou2@hanyang.ac.kr

**ABSTRACT**

**Background:** In our previous study, PLR was shown to inhibit prion propagation in the cell culture model of prion diseases (1). In current study, we investigated the effect of PLR *in vivo* whether it inhibits prions.

**Material and Methods:** Firstly, mice inoculated intraperitoneally (IP) with prions were administered with PLR twice a week for 4 weeks 10-40 mg/kg by the IP route. Spleen were collected from infected mice at every 21-day until 84 days post-inoculation. In another approach, to observe the delay of disease onset, mice infected intracerebrally with prions, were administered with PLR twice a week for 4 weeks 10-25 mg/kg PLR by the IP route. As disease onsets, the brain and spleen were collected. The level of PrP<sup>Sc</sup> in the brain and spleen were detected by Western blotting. The accumulation of the PrP<sup>Sc</sup> and glial fibrillary acidic protein were monitored using immunohistochemistry. Vacuolation in the brain was investigated using H&E stained brain sections.

**Result:** PrP<sup>Sc</sup> propagation was suppressed in the spleen of PLR-treated group during the asymptomatic stage. Administration with 10-25 mg/kg PLR prolonged the incubation time of prion diseases over seven days. At the disease onset, the level of PrP<sup>Sc</sup> reduced in the spleen, but unchanged in the brain. However, immunohistochemistry analysis showed that the level of PrP<sup>Sc</sup> in white pulp of the spleen and specific areas of the brain, such as hypothalamus, cortex, and hippocampus, but not mid-brain, thalamus, and straitum, was reduced by PLR. Moreover, glial fibrillary acidic protein staining indicated that PLR decreased astrogliosis in thalamus, cortex, and polymorph layer 3 of cerebral cortex of brain but not in other brain regions. In addition, H & E staining indicated that the decrease of vacuolation area and vacuole counts in mid-brain, hypothalamus, hippocampus, and straitum regions of the brains from the PLR-administered mice.

**Conclusion:** Considering suppression of prion propagation in spleen during asymptomatic stage, significant
<figure><img src="image_61.png" /></figure>

N96S<sub>Sh</sub>/S169N<sub>Cer</sub>/N173T<sub>Cer</sub> was shortened by 11.5 h and 14.0 h compared to rMoPrP. Taken together, the inhibitory factors may also interfere with the region including CerPrP specific N173, and T177.

**Conclusion:** Our results suggest that conversion efficiency in the RT-QuIC reaction may be interfered with by the inhibitory factors at least at two regions of the PrP molecule, one is the C-terminus and the other is the region including CerPrP-specific aa 173 N and 177 T. These regions have been shown to be adjacent in the three-dimensional structure of PrP<sup>C</sup>; therefore, inhibitory factors may bind to the site including these regions.

**91. PrP regulates neural stem cell senescence through epidermal growth factor receptor**

Bradley R. Groveman, Simote T. Foliaki, Aleksandar R. Wood, Brent Race and Cathryn L. Haigh
Rocky Mountain Laboratories, NIAID, NIH, Hamilton, MT, USA

CONTACT Bradley R. Groveman Bradley.groveman@nih.gov

**ABSTRACT**

The prion protein (PrP) is expressed in a wide variety of progenitor cells and is known to be involved in neural stem cell self-renewal processes. The mechanisms by which PrP acts to modulate cell homeostasis have, however, not been fully elucidated. Recent studies have shown that PrP has a direct role in preventing premature senescence [1], and that knockdown of PrP increases senescence markers and decreases the cell proliferative potential. PrP has also been shown to regulate epidermal growth factor receptor (EGFR) through Notch signalling. Notch signalling, which can upregulate EGFR, is disrupted in the absence of PrP [2], leading to a reduction in EGFR expression. Furthermore, inhibition of EGFR or depletion of EGF have been shown to suppress senescence in proliferative cells [3]. Herein we attempt to show the role of PrP in regulating cellular senescence through the EGFR pathway.

**KEYWORDS:** Prion; PrP; neural stem cell; senescence; epidermal growth factor receptor; EGFR

**References**

[1] Boilan E, et al. Mech Ageing Dev. 2018;170:106-113.

[2] Martin-Lannerée S, et al. Stem Cells. 2017;35(3):754-765.

[3] Alexander PB, et al. Cell Res. 2015;25(1):135-138.

**92. Region-specific interference of PrP<sup>Sc</sup> accumulation in spleen and brain by poly-L-arginine (PLR)**

Jieun Kim<sup>a</sup>, Taeyeon Kim<sup>b</sup>, A-ran Kim<sup>a</sup>, Hakmin Lee<sup>a</sup>, Trang H. T. Trinh<sup>a</sup>, Junwu Shin<sup>a</sup>, Sungeun Lee<sup>a</sup> and Chongsuk Ryou<sup>a</sup>

<sup>a</sup>Department of Pharmacy, College of Pharmacy and Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan, Gyeonggi-do, Republic of Korea; <sup>b</sup>Division of Developmental Biology and Physiology, School of Biosciences and Chemistry, Institute for Basic science, Sungshin University, Seoul, Republic of Korea.

CONTACT Chongsuk Ryou cryou2@hanyang.ac.kr

**ABSTRACT**

**Background:** In our previous study, PLR was shown to inhibit prion propagation in the cell culture model of prion diseases (1). In current study, we investigated the effect of PLR *in vivo* whether it inhibits prions.

**Material and Methods:** Firstly, mice inoculated intraperitoneally (IP) with prions were administered with PLR twice a week for 4 weeks 10-40 mg/kg by the IP route. Spleen were collected from infected mice at every 21-day until 84 days post-inoculation. In another approach, to observe the delay of disease onset, mice infected intracerebrally with prions, were administered with PLR twice a week for 4 weeks 10-25 mg/kg PLR by the IP route. As disease onsets, the brain and spleen were collected. The level of PrP<sup>Sc</sup> in the brain and spleen were detected by Western blotting. The accumulation of the PrP<sup>Sc</sup> and glial fibrillary acidic protein were monitored using immunohistochemistry. Vacuolation in the brain was investigated using H&E stained brain sections.

**Result:** PrP<sup>Sc</sup> propagation was suppressed in the spleen of PLR-treated group during the asymptomatic stage. Administration with 10-25 mg/kg PLR prolonged the incubation time of prion diseases over seven days. At the disease onset, the level of PrP<sup>Sc</sup> reduced in the spleen, but unchanged in the brain. However, immunohistochemistry analysis showed that the level of PrP<sup>Sc</sup> in white pulp of the spleen and specific areas of the brain, such as hypothalamus, cortex, and hippocampus, but not mid-brain, thalamus, and straitum, was reduced by PLR. Moreover, glial fibrillary acidic protein staining indicated that PLR decreased astrogliosis in thalamus, cortex, and polymorph layer 3 of cerebral cortex of brain but not in other brain regions. In addition, H & E staining indicated that the decrease of vacuolation area and vacuole counts in mid-brain, hypothalamus, hippocampus, and straitum regions of the brains from the PLR-administered mice.

**Conclusion:** Considering suppression of prion propagation in spleen during asymptomatic stage, significant
<figure><img src="image_58.png" /></figure>

increase of survival time and brain region-specific inhibition of neuropathological onsets, we demonstrated PLR as a potential therapeutic candidate for prion diseases and the value of further studies.

**KEYWORDS:** Prion; poly-L-arginine; PrPSc; spleen; brain

## Reference

[1] Waqas, et al. Mol Cell Biochem. 2017;428(1-2):57-66.

## 93. Prion protein distribution among white-tailed deer and mule deer exocrine glands

Anthony M. Ness<sup>a,b</sup>, Kelsey Saboraki<sup>c</sup>, Camilo D. Velásquez<sup>a,b</sup>, Danielle Gushue<sup>a,b</sup>, Alicia O. Garcia<sup>a,b</sup>, Joshua Craig<sup>a,b</sup>, Taraneh Ashrafi<sup>a,d</sup>, Judd Aiken<sup>a,d</sup>, Susan Lingle<sup>c*</sup>, Debbie McKenzie<sup>1,2*</sup>

<sup>a</sup>Centre for Prions and Protein Folding Diseases, University of Alberta;

<sup>b</sup>Department of Biological Sciences, University of Alberta;

<sup>c</sup>Department of Biology, University of Winnipeg; <sup>d</sup>Department of Agricultural, Food, and Nutritional Sciences, University of Alberta

CONTACT Anthony M. Ness amness@ualberta.ca

## ABSTRACT

**Background:** Chronic wasting disease (CWD) is a contagious neurodegenerative prion disease afflicting cervids in Canada, the United States, South Korea, and Scandinavia. The precise mechanism of natural CWD transmission is poorly understood but is believed to involve both direct contact between cervids and exposure to environmental reservoirs of PrP<sup>CWD</sup>. The scent glands in the face and legs of deer are numerous and serve for direct and indirect inter-individual communication and social behaviours. Glandular innervation by the autonomic nervous system may act as a site for CWD prion uptake, neuroinvasion, and retrograde transport towards the central nervous system.

**Materials and Methods:** Facial and leg exocrine glands were collected from adult and white-tailed deer and mule deer males and females harvested from Canadian Forces Base Wainright, Alberta, Canada. Vomeronasal, forehead, pre-orbital, parotid, and nasal glands in the head, and the tarsal, metatarsal, and interdigital glands in the legs were homogenized for biochemical analysis or embedded for histological analysis. White-tailed deer and mule deer glandular PrP<sup>C</sup> levels were determined by Western blot and ELISA. The distribution of PrP<sup>D</sup> was evaluated by immunohistochemistry (IHC).

**Results and Discussion:** We show the relative PrP<sup>C</sup> levels and distribution in the various facial and leg exocrine glands studied. IHC revealed PrP<sup>D</sup> within select glands. The involvement of cervid exocrine

glands in CWD transmission has been poorly studied; however, our observations suggest these glands act as sites for prion entry and shedding – deserving further investigation. Cervid exocrine glands may represent a biologically and ecologically important transmission route of CWD in deer. Further, concurrent research suggests that species-and sex-specific social behaviours, involving the glandular tissues, may provide insights as to why CWD is most prevalent in mule deer males and least prevalent in white-tailed females.

**KEYWORDS:** Chronic wasting disease; prion; deer; cervids; glands; transmission

## 94. Plasma tau and neurofilament light chain as biomarkers for the human prion diseases: evaluation in a large prospective natural history cohort

Andrew G. B. Thompson<sup>a</sup>, Prodromos Anastasiadis<sup>a</sup>, Akin Nihat<sup>a,b</sup>, Tze Mok How<sup>a,b</sup>, Peter Rudge<sup>a,b</sup>, Amanda Heslegrave<sup>c</sup>, Henrik Zetterberg<sup>c</sup>, John Collinge<sup>a,b</sup>, Graham S. Jackson<sup>a</sup> and Simon H. Mead<sup>a,b</sup>

<sup>a</sup>MRC Prion Unit, University College London, UK; <sup>b</sup>National Prion Clinic, University College London Hospitals NHS Foundation Trust, UK; <sup>c</sup>Dementia Research Institute Biomarkers Laboratory, University College London, UK.

CONTACT Andrew GB Thompson

## ABSTRACT

**Background:** Diagnostic methods for prion disease have advanced substantially, but unmet needs remain for biomarkers to fill a number of other important roles. These include monitoring of disease activity, predicting symptom onset in individuals at high risk (such as carriers of prion protein gene (PRNP) mutations), and enabling earlier diagnosis. Tau and neurofilament light chain (NfL) protein concentrations in blood are known to be elevated in prion disease, and have shown promise as quantitative markers of central nervous system disease activity in a range of other conditions.

**Materials and Methods:** We used ultrasensitive immuno-assays (Simoa) to measure tau and NfL concentrations in 709 plasma samples taken from 377 individuals with prion disease during their participation in a large prospective clinical study (the UK National Prion Monitoring Cohort), in addition to healthy and neurological control groups. This provides an unprecedented opportunity to analyse their clinical associations and potential as biomarkers.

**Results:** Plasma tau and NfL concentrations are increased across all prion disease types. For distinguishing sporadic CJD (sCJD) from a clinically relevant
<figure><img src="image_62.png" /></figure>

increase of survival time and brain region-specific inhibition of neuropathological onsets, we demonstrated PLR as a potential therapeutic candidate for prion diseases and the value of further studies.

**KEYWORDS:** Prion; poly-L-arginine; PrPSc; spleen; brain

## Reference

[1] Waqas, et al. Mol Cell Biochem. 2017;428(1-2):57-66.

## 93. Prion protein distribution among white-tailed deer and mule deer exocrine glands

Anthony M. Ness<sup>a,b</sup>, Kelsey Saboraki<sup>c</sup>, Camilo D. Velásquez<sup>a,b</sup>, Danielle Gushue<sup>a,b</sup>, Alicia O. Garcia<sup>a,b</sup>, Joshua Craig<sup>a,b</sup>, Taraneh Ashrafi<sup>a,d</sup>, Judd Aiken<sup>a,d</sup>, Susan Lingle<sup>c*</sup>, Debbie McKenzie<sup>1,2*</sup>

CONTACT Anthony M. Ness amness@ualberta.ca

## ABSTRACT

**Background:** Chronic wasting disease (CWD) is a contagious neurodegenerative prion disease afflicting cervids in Canada, the United States, South Korea, and Scandinavia. The precise mechanism of natural CWD transmission is poorly understood but is believed to involve both direct contact between cervids and exposure to environmental reservoirs of PrP<sup>CWD</sup>. The scent glands in the face and legs of deer are numerous and serve for direct and indirect inter-individual communication and social behaviours. Glandular innervation by the autonomic nervous system may act as a site for CWD prion uptake, neuroinvasion, and retrograde transport towards the central nervous system.

**Materials and Methods:** Facial and leg exocrine glands were collected from adult and white-tailed deer and mule deer males and females harvested from Canadian Forces Base Wainright, Alberta, Canada. Vomeronasal, forehead, pre-orbital, parotid, and nasal glands in the head, and the tarsal, metatarsal, and interdigital glands in the legs were homogenized for biochemical analysis or embedded for histological analysis. White-tailed deer and mule deer glandular PrP<sup>C</sup> levels were determined by Western blot and ELISA. The distribution of PrP<sup>D</sup> was evaluated by immunohistochemistry (IHC).

**Results and Discussion:** We show the relative PrP<sup>C</sup> levels and distribution in the various facial and leg exocrine glands studied. IHC revealed PrP<sup>D</sup> within select glands. The involvement of cervid exocrine

glands in CWD transmission has been poorly studied; however, our observations suggest these glands act as sites for prion entry and shedding – deserving further investigation. Cervid exocrine glands may represent a biologically and ecologically important transmission route of CWD in deer. Further, concurrent research suggests that species-and sex-specific social behaviours, involving the glandular tissues, may provide insights as to why CWD is most prevalent in mule deer males and least prevalent in white-tailed females.

**KEYWORDS:** Chronic wasting disease; prion; deer; cervids; glands; transmission

## 94. Plasma tau and neurofilament light chain as biomarkers for the human prion diseases: evaluation in a large prospective natural history cohort

Andrew G. B. Thompson<sup>a</sup>, Prodromos Anastasiadis<sup>a</sup>, Akin Nihat<sup>a,b</sup>, Tze Mok How<sup>a,b</sup>, Peter Rudge<sup>a,b</sup>, Amanda Heslegrave<sup>c</sup>, Henrik Zetterberg<sup>c</sup>, John Collinge<sup>a,b</sup>, Graham S. Jackson<sup>a</sup> and Simon H. Mead<sup>a,b</sup>

CONTACT Andrew GB Thompson

## ABSTRACT

**Background:** Diagnostic methods for prion disease have advanced substantially, but unmet needs remain for biomarkers to fill a number of other important roles. These include monitoring of disease activity, predicting symptom onset in individuals at high risk (such as carriers of prion protein gene (PRNP) mutations), and enabling earlier diagnosis. Tau and neurofilament light chain (NfL) protein concentrations in blood are known to be elevated in prion disease, and have shown promise as quantitative markers of central nervous system disease activity in a range of other conditions.

**Materials and Methods:** We used ultrasensitive immuno-assays (Simoa) to measure tau and NfL concentrations in 709 plasma samples taken from 377 individuals with prion disease during their participation in a large prospective clinical study (the UK National Prion Monitoring Cohort), in addition to healthy and neurological control groups. This provides an unprecedented opportunity to analyse their clinical associations and potential as biomarkers.

**Results:** Plasma tau and NfL concentrations are increased across all prion disease types. For distinguishing sporadic CJD (sCJD) from a clinically relevant

<footnote><sup>a</sup>Centre for Prions and Protein Folding Diseases, University of Alberta;</footnote>

<footnote><sup>b</sup>Department of Biological Sciences, University of Alberta;</footnote>

<footnote><sup>c</sup>Department of Biology, University of Winnipeg; <sup>d</sup>Department of Agricultural, Food, and Nutritional Sciences, University of Alberta</footnote>

<footnote><sup>a</sup>MRC Prion Unit, University College London, UK; <sup>b</sup>National Prion Clinic, University College London Hospitals NHS Foundation Trust, UK; <sup>c</sup>Dementia Research Institute Biomarkers Laboratory, University College London, UK.</footnote>
<figure><img src="image_59.png" /></figure>

‘prion mimic’ control group, both show moderate diag-
nostic value (area under ROC curves: tau 0.81, NfL
0.72). In sCJD, plasma NfL was substantially elevated
in every sample tested, including during early disease
with minimal functional impairment. Plasma tau was
independently associated with *rate* of clinical progres-
sion in sCJD, while plasma NfL showed independent
association with *severity* of functional impairment. In
asymptomatic PRNP mutation carriers, plasma NfL was
higher in samples taken within 2 years of symptom
onset than in those taken earlier. We present biomarker
trajectories for 9 PRNP mutation carriers who devel-
oped symptoms during follow-up, and show potential
for plasma NfL as a ‘proximity marker’.

**Conclusions:** We conclude that plasma tau and NfL represent the best currently available candidates for a number of vital biomarker roles in prion disease. We discuss directions for future work, particularly focussing on ways that these biomarkers could be valuable for therapeutic research.

95. Gene-edited murine cell lines for propagation of chronic wasting disease prions

Rupali Walia<sup>a,b</sup>, Rohit Chandra<sup>a,c</sup> and Hermann M. Schatzl<sup>a,b</sup>

CONTACT Rohit Chandra rohit.chandra@ucalgary.ca

ABSTRACT

Chronic wasting disease (CWD), a highly contagious prion
disease, has recently emerged as a threat to both farmed
and free ranging cervids across North America, South
Korea, and Europe. There is thus a pressing need to design
versatile cell culture models which can complement the
animal studies on CWD and help to contain this disease.
Defining such cell culture models is mostly a random
process, and includes extensive subcloning and selection.
To extend the range of cell models propagating CWD
prions, we gene-edited mouse cell lines known to well
propagate murine prions. Endogenous prion protein
(PrP) was ablated in CAD5 cells and immortalized mouse
embryonic fibroblasts (MEF), using CRISPR-Cas9 gene
editing. PrP knock-out cells were reconstituted with
mouse, bank vole (BV) and cervid PrP genes by lentiviral
transduction. Reconstituted cells expressing mouse PrP
provided proof-of-concept for re-established prion infec-
tion. The promiscuous nature of BV PrP<sup>c</sup> was exploited to
demonstrate the permissiveness of these reconstituted cell

lines to infection by CWD prions, even without subclon-
ing. Cells reconstituted with BV or cervid PrP and infected
with two types of CWD prions, white-tailed deer and mule
deer prions, tested positive in prion conversion assay,
whereas non-reconstituted cells were negative. We here
provide a successful strategy for designing cell lines with
specific as well as broader susceptibility to prion infection,
while retaining a common host background. Data obtained
by infecting such gene-targeted and re-constituted cultured
cells will provide important new insights into species and
prion strain barriers and provide new experimental sys-
tems for studying CWD prions *in vitro*.

96. PrPSc aggregation state dictates biochemical
properties of prions and disease pathogenesis

Sheng Chun Chang<sup>a</sup>, Samia Hannaoui<sup>a</sup>, Stefanie
Czub<sup>b</sup> and Sabine Gilch<sup>a</sup>

CONTACT Sheng Chun Chang shengchun.chang@ucalgary.ca

ABSTRACT

**Background:** Transmissible spongiform encephalopathies (TSE) are devastating neurodegenerative diseases caused by the conversion of the host-encoded cellular prion protein (PrP<sup>C</sup>) into the pathological misfolded isoform known as PrP<sup>Sc</sup>. Various strains of prions exist, each of which consists of a unique conformational, biochemical, and biological profile. Previous studies have indicated that the profile of PrP<sup>Sc</sup> aggregation states also is strain specific. Our study investigates whether biochemical and biological properties of individual fractions of PrP<sup>Sc</sup> are stable upon serial passage *in vivo*.

**Methods:** A pool of CWD2 elk brain homogenate was subject to a sedimentation velocity gradient and separated into 30 fractions. Alternating fractions were intracerebrally inoculated into transgenic mice overexpressing elk PrP<sup>C</sup> (tgElk) and incubation times and clinical signs were monitored. Brains (first passage) were harvested upon the progression of terminal prion disease. Brains of three mice each inoculated with PrP<sup>Sc</sup> fractions 2–6 (low molecular weight (MW)), 8–18 (medium MW), and 20–30 (high MW), respectively, were used for second passage into tgElk mice. Brain samples were subjected to digestion with increasing concentrations of Proteinase K (PK) to determine the PK concentration required to degrade 50% of PrP<sup>Sc</sup> (cPK<sub>50</sub>).

**Results:** In the first passage, mice inoculated with low MW PrP<sup>Sc</sup> aggregates exhibited symptoms of hyper-

<footnote><sup>a</sup>Department of Comparative Biology & Experimental Medicine, University of Calgary, Calgary, Canada; <sup>b</sup>Calgary Prion Research Unit, University of Calgary, Calgary, Canada; <sup>c</sup>Faculty of Science, University of British Columbia, Vancouver</footnote>

<footnote><sup>a</sup>Department of Ecosystem and Public Health, Faculty of Veterinary Medicine, Calgary Prion Research Unit, Hotchkiss Brain Institute, University of Calgary, Calgary, Canada; <sup>b</sup>Canadian Food Inspection Agency Lethbridge Laboratories, Lethbridge, Canada</footnote>
<figure><img src="image_63.png" /></figure>

‘prion mimic’ control group, both show moderate diagnostic value (area under ROC curves: tau 0.81, NfL 0.72). In sCJD, plasma NfL was substantially elevated in every sample tested, including during early disease with minimal functional impairment. Plasma tau was independently associated with *rate* of clinical progression in sCJD, while plasma NfL showed independent association with *severity* of functional impairment. In asymptomatic PRNP mutation carriers, plasma NfL was higher in samples taken within 2 years of symptom onset than in those taken earlier. We present biomarker trajectories for 9 PRNP mutation carriers who developed symptoms during follow-up, and show potential for plasma NfL as a ‘proximity marker’.

**Conclusions:** We conclude that plasma tau and NfL represent the best currently available candidates for a number of vital biomarker roles in prion disease. We discuss directions for future work, particularly focussing on ways that these biomarkers could be valuable for therapeutic research.

## 95. Gene-edited murine cell lines for propagation of chronic wasting disease prions

Rupali Walia<sup>a,b</sup>, Rohit Chandra<sup>a,c</sup> and Hermann M. Schatzl<sup>a,b</sup>

CONTACT Rohit Chandra rohit.chandra@ucalgary.ca

### ABSTRACT

Chronic wasting disease (CWD), a highly contagious prion disease, has recently emerged as a threat to both farmed and free ranging cervids across North America, South Korea, and Europe. There is thus a pressing need to design versatile cell culture models which can complement the animal studies on CWD and help to contain this disease. Defining such cell culture models is mostly a random process, and includes extensive subcloning and selection. To extend the range of cell models propagating CWD prions, we gene-edited mouse cell lines known to well propagate murine prions. Endogenous prion protein (PrP) was ablated in CAD5 cells and immortalized mouse embryonic fibroblasts (MEF), using CRISPR-Cas9 gene editing. PrP knock-out cells were reconstituted with mouse, bank vole (BV) and cervid PrP genes by lentiviral transduction. Reconstituted cells expressing mouse PrP provided proof-of-concept for re-established prion infection. The promiscuous nature of BV PrP<sup>c</sup> was exploited to demonstrate the permissiveness of these reconstituted cell

lines to infection by CWD prions, even without subcloning. Cells reconstituted with BV or cervid PrP and infected with two types of CWD prions, white-tailed deer and mule deer prions, tested positive in prion conversion assay, whereas non-reconstituted cells were negative. We here provide a successful strategy for designing cell lines with specific as well as broader susceptibility to prion infection, while retaining a common host background. Data obtained by infecting such gene-targeted and re-constituted cultured cells will provide important new insights into species and prion strain barriers and provide new experimental systems for studying CWD prions *in vitro*.

## 96. PrPSc aggregation state dictates biochemical properties of prions and disease pathogenesis

Sheng Chun Chang<sup>a</sup>, Samia Hannaoui<sup>a</sup>, Stefanie Czub<sup>b</sup> and Sabine Gilch<sup>a</sup>

CONTACT Sheng Chun Chang shengchun.chang@ucalgary.ca

### ABSTRACT

**Background:** Transmissible spongiform encephalopathies (TSE) are devastating neurodegenerative diseases caused by the conversion of the host-encoded cellular prion protein (PrP<sup>C</sup>) into the pathological misfolded isoform known as PrP<sup>Sc</sup>. Various strains of prions exist, each of which consists of a unique conformational, biochemical, and biological profile. Previous studies have indicated that the profile of PrP<sup>Sc</sup> aggregation states also is strain specific. Our study investigates whether biochemical and biological properties of individual fractions of PrP<sup>Sc</sup> are stable upon serial passage *in vivo*.

**Methods:** A pool of CWD2 elk brain homogenate was subject to a sedimentation velocity gradient and separated into 30 fractions. Alternating fractions were intracerebrally inoculated into transgenic mice overexpressing elk PrP<sup>C</sup> (tgElk) and incubation times and clinical signs were monitored. Brains (first passage) were harvested upon the progression of terminal prion disease. Brains of three mice each inoculated with PrP<sup>Sc</sup> fractions 2–6 (low molecular weight (MW)), 8–18 (medium MW), and 20–30 (high MW), respectively, were used for second passage into tgElk mice. Brain samples were subjected to digestion with increasing concentrations of Proteinase K (PK) to determine the PK concentration required to degrade 50% of PrP<sup>Sc</sup> (cPK<sub>50</sub>).

**Results:** In the first passage, mice inoculated with low MW PrP<sup>Sc</sup> aggregates exhibited symptoms of hyper-

<footnote><sup>a</sup>Department of Comparative Biology & Experimental Medicine, University of Calgary, Calgary, Canada; <sup>b</sup>Calgary Prion Research Unit, University of Calgary, Calgary, Canada; <sup>c</sup>Faculty of Science, University of British Columbia, Vancouver</footnote>

<footnote><sup>a</sup>Department of Ecosystem and Public Health, Faculty of Veterinary Medicine, Calgary Prion Research Unit, Hotchkiss Brain Institute, University of Calgary, Calgary, Canada; <sup>b</sup>Canadian Food Inspection Agency Lethbridge Laboratories, Lethbridge, Canada</footnote>
<figure><img src="image_60.png" /></figure>

excitability, while those inoculated with high MW aggregates were lethargic and gained weight, and a prolonged incubation was observed in both groups, while those given medium MW aggregates had a decreased incubation time. Biochemical analysis showed that mice inoculated with low or medium MW aggregates had $Pr^{PSc}$ that were markedly less resistant to PK digestion compared to the $Pr^{PSc}$ from mice inoculated with high MW aggregates. These characteristics were no longer observed in the second passage, where the incubation period and the PK resistance among the groups were similar, though the observed symptoms in the mice inoculated with the low or high MW $Pr^{PSc}$ were still hyper-excitability and lethargy, respectively, as in the first passage.

**Conclusion:** Our study demonstrates that inoculation of different $Pr^{PSc}$ aggregation states separated by molecular weight can lead to differing pathogenesis and transient changes to the biochemical characteristics. This will add novel knowledge to our understanding of transmissible biochemical characteristics of prions.

**97. In vitro seeding activity of glycoform-deficient prions from variably protease-sensitive prionopathy and familial CJD associated with $Pr^{V180I}$ mutation**

Zerui Wang<sup>a,b</sup>, Jue Yuan<sup>a</sup>, Pingping Shen<sup>a,b</sup>, Romany Abskharon<sup>c</sup>, Yue Lang<sup>a,b</sup>, Johnny Dang<sup>a</sup>, Alise Adornato<sup>a</sup>, Ling Xu<sup>a</sup>, Jiafeng Chen<sup>b</sup>, Jiachun Feng<sup>b</sup>, Mohammed Moudjou<sup>d</sup>, Tetsuyuki Kitamoto<sup>e</sup>, Jan Langeveld<sup>f</sup>, Brian Appleby<sup>a,g,h</sup>, Jiyan Ma<sup>c</sup>, Qingzhong Kong<sup>a,g,h</sup>, Robert B. Petersen<sup>a,i</sup>, Li Cui<sup>b</sup> and Wen-Quan Zou<sup>a,b,g,h</sup>

CONTACT Wen-Quan Zou WQZ wxz6@case.edu

**ABSTRACT**

Both sporadic variably protease-sensitive prionopathy (VPSPr) and familial Creutzfeldt-Jakob disease linked to the prion protein (PrP) V180I mutation

($CJD^{V180I}$) have been found to share a unique pathological prion protein ($Pr^{PSc}$) pattern that lacks the protease-resistant $Pr^{PSc}$ glycosylated at residue 181, apparently because two of four cellular PrP ($Pr^{Pc}$) glycoforms are not converted into $Pr^{PSc}$. To investigate the seeding activity of these unique $Pr^{PSc}$ molecules, we conducted *in vitro* prion conversion experiments using serial protein misfolding cyclic amplification (sPMCA) and real-time quaking-induced conversion (RT-QuIC) assays with different $Pr^{Pc}$ substrates. Unexpectedly, we observed that the seeding of $Pr^{PSc}$ from VPSPr or $CJD^{V180I}$ in the sPMCA reaction with brain homogenates from normal human or humanized transgenic (Tg) mice generated $Pr^{PSc}$ molecules that are dominated by the diglycosylated isoform, along with $Pr^{PSc}$ monoglycosylated at residue 181. The efficiency of $Pr^{PSc}$ amplification was significantly higher in MM than in VV human brain homogenate, whereas it was higher in TgVV than in TgMM mouse brain homogenate. $Pr^{Pc}$ from the brain homogenate mixture of TgMM and Tg mice expressing $Pr^{V180I}$ mutation ($Tg180$), but not that from $TgV180I$ alone, was converted into $Pr^{PSc}$ by seeding with the VPSPr or $CJD^{V180I}$. The RT-QuIC seeding activity of $Pr^{PSc}$ from VPSPr and $CJD^{V180I}$ was significantly lower than that of sCJD. Our results suggest that the formation of glycoform-selective prions may be associated with an unidentified factor in the affected brain and the glycoform-deficiency of $Pr^{PSc}$ does not affect the glycoforms of *in vitro* newly-amplified $Pr^{PSc}$

**Funding**

Supported in part by the CJD Foundation and the National Institutes of Health (NIH) NS062787 and NS087588 to W.Q.Z., NS062787 and NS109532 to W. Q.Z., and Q.K., NS088604 to Q.K., the Centers for Disease Control and Prevention Contract UR8/CCU515004 to B.S.A., the National Natural Science Foundation of China (NNSFC) [No. 81,801,207] to PS, as well as NNSFC [No. 81,671,186] to LC.

**98. Longitudinal comparison of real-time conversion and immunohistochemistry for detection of chronic wasting disease in orally exposed white-tailed deer**

Nathaniel D. Denkers<sup>a</sup>, Davin M. Henderson<sup>a</sup>, Clare E. Hoover<sup>a,b*</sup>, Amy V. Nalls<sup>a</sup>, Erin McNulty<sup>a</sup>, Candace K. Mathiason<sup>a</sup> and Edward A. Hoover<sup>a</sup>

<footnote><sup>a</sup>Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA; <sup>b</sup>Department of Neurology, The First Hospital of Jilin University, Changchun, Jilin Province, the People's Republic of China; <sup>c</sup>Center for Neurodegenerative Science, Van Andel Research Institute, Grand Rapids, MI, USA; <sup>d</sup>NRA, Université Paris-Saclay, UR892, Virologie Immunologie Moléculaires, Jouy-en-Josas, France; <sup>e</sup>Center for Prion Diseases, Tohoku University Graduate School of Medicine, Sendai, Japan; <sup>f</sup>Wageningen BioVeterinary Research, Lelystad, the Netherlands; <sup>g</sup>National Prion Disease Pathology Surveillance Center, Case Western Reserve University School of Medicine, Cleveland, OH, USA; <sup>h</sup>Department of Neurology, Case Western Reserve University School of Medicine, Cleveland, OH, USA; <sup>i</sup>Foundation Sciences, Central Michigan University College of Medicine, Mount Pleasant, MI, USA</footnote>
<figure><img src="image_64.png" /></figure>

excitability, while those inoculated with high MW aggregates were lethargic and gained weight, and a prolonged incubation was observed in both groups, while those given medium MW aggregates had a decreased incubation time. Biochemical analysis showed that mice inoculated with low or medium MW aggregates had $Pr^{PSc}$ that were markedly less resistant to PK digestion compared to the $Pr^{PSc}$ from mice inoculated with high MW aggregates. These characteristics were no longer observed in the second passage, where the incubation period and the PK resistance among the groups were similar, though the observed symptoms in the mice inoculated with the low or high MW $Pr^{PSc}$ were still hyper-excitability and lethargy, respectively, as in the first passage.

**Conclusion:** Our study demonstrates that inoculation of different $Pr^{PSc}$ aggregation states separated by molecular weight can lead to differing pathogenesis and transient changes to the biochemical characteristics. This will add novel knowledge to our understanding of transmissible biochemical characteristics of prions.

**97. In vitro seeding activity of glycoform-deficient prions from variably protease-sensitive prionopathy and familial CJD associated with $Pr^{V180I}$ mutation**

Zerui Wang<sup>a,b</sup>, Jue Yuan<sup>a</sup>, Pingping Shen<sup>a,b</sup>, Romany Abskharon<sup>c</sup>, Yue Lang<sup>a,b</sup>, Johnny Dang<sup>a</sup>, Alise Adornato<sup>a</sup>, Ling Xu<sup>a</sup>, Jiafeng Chen<sup>b</sup>, Jiachun Feng<sup>b</sup>, Mohammed Moudjou<sup>d</sup>, Tetsuyuki Kitamoto<sup>e</sup>, Jan Langeveld<sup>f</sup>, Brian Appleby<sup>a,g,h</sup>, Jiyan Ma<sup>c</sup>, Qingzhong Kong<sup>a,g,h</sup>, Robert B. Petersen<sup>a,i</sup>, Li Cui<sup>b</sup> and Wen-Quan Zou<sup>a,b,g,h</sup>

CONTACT Wen-Quan Zou WQZ wxz6@case.edu

**ABSTRACT**

Both sporadic variably protease-sensitive prionopathy (VPSPr) and familial Creutzfeldt-Jakob disease linked to the prion protein (PrP) V180I mutation

($CJD^{V180I}$) have been found to share a unique pathological prion protein ($Pr^{PSc}$) pattern that lacks the protease-resistant $Pr^{PSc}$ glycosylated at residue 181, apparently because two of four cellular PrP ($Pr^{Pc}$) glycoforms are not converted into $Pr^{PSc}$. To investigate the seeding activity of these unique $Pr^{PSc}$ molecules, we conducted *in vitro* prion conversion experiments using serial protein misfolding cyclic amplification (sPMCA) and real-time quaking-induced conversion (RT-QuIC) assays with different $Pr^{Pc}$ substrates. Unexpectedly, we observed that the seeding of $Pr^{PSc}$ from VPSPr or $CJD^{V180I}$ in the sPMCA reaction with brain homogenates from normal human or humanized transgenic (Tg) mice generated $Pr^{PSc}$ molecules that are dominated by the diglycosylated isoform, along with $Pr^{PSc}$ monoglycosylated at residue 181. The efficiency of $Pr^{PSc}$ amplification was significantly higher in MM than in VV human brain homogenate, whereas it was higher in TgVV than in TgMM mouse brain homogenate. $Pr^{Pc}$ from the brain homogenate mixture of TgMM and Tg mice expressing $Pr^{V180I}$ mutation ($Tg180$), but not that from $TgV180I$ alone, was converted into $Pr^{PSc}$ by seeding with the VPSPr or $CJD^{V180I}$. The RT-QuIC seeding activity of $Pr^{PSc}$ from VPSPr and $CJD^{V180I}$ was significantly lower than that of sCJD. Our results suggest that the formation of glycoform-selective prions may be associated with an unidentified factor in the affected brain and the glycoform-deficiency of $Pr^{PSc}$ does not affect the glycoforms of *in vitro* newly-amplified $Pr^{PSc}$

**Funding**

Supported in part by the CJD Foundation and the National Institutes of Health (NIH) NS062787 and NS087588 to W.Q.Z., NS062787 and NS109532 to W. Q.Z., and Q.K., NS088604 to Q.K., the Centers for Disease Control and Prevention Contract UR8/CCU515004 to B.S.A., the National Natural Science Foundation of China (NNSFC) [No. 81,801,207] to PS, as well as NNSFC [No. 81,671,186] to LC.

**98. Longitudinal comparison of real-time conversion and immunohistochemistry for detection of chronic wasting disease in orally exposed white-tailed deer**

Nathaniel D. Denkers<sup>a</sup>, Davin M. Henderson<sup>a</sup>, Clare E. Hoover<sup>a,b*</sup>, Amy V. Nalls<sup>a</sup>, Erin McNulty<sup>a</sup>, Candace K. Mathiason<sup>a</sup> and Edward A. Hoover<sup>a</sup>

<footnote><sup>a</sup>Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA; <sup>b</sup>Department of Neurology, The First Hospital of Jilin University, Changchun, Jilin Province, the People's Republic of China; <sup>c</sup>Center for Neurodegenerative Science, Van Andel Research Institute, Grand Rapids, MI, USA; <sup>d</sup>NRA, Université Paris-Saclay, UR892, Virologie Immunologie Moléculaires, Jouy-en-Josas, France; <sup>e</sup>Center for Prion Diseases, Tohoku University Graduate School of Medicine, Sendai, Japan; <sup>f</sup>Wageningen BioVeterinary Research, Lelystad, the Netherlands; <sup>g</sup>National Prion Disease Pathology Surveillance Center, Case Western Reserve University School of Medicine, Cleveland, OH, USA; <sup>h</sup>Department of Neurology, Case Western Reserve University School of Medicine, Cleveland, OH, USA; <sup>i</sup>Foundation Sciences, Central Michigan University College of Medicine, Mount Pleasant, MI, USA</footnote>
<figure><img src="image_61.png" /></figure>

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

CONTACT Nathaniel D. Denkers nathaniel.denkers@colostate.edu

*Present address: AstraZeneca, Waltham, NJ

## ABSTRACT

**Background:** Chronic wasting disease (CWD) continues to expand across North America and Canada, and more recently was discovered in Scandinavia. While the exact mechanism of CWD transmission has yet to be elucidated, early diagnosis of infected animals remains a priority in curtailing its spread. While immuno-histochemistry (IHC) and enzyme linked immunosorbent assay (ELISA) remain the gold standards for diagnosing CWD, real-time quaking induced conversion (RT-QuIC) is capable of detecting substantially lower concentrations of prions, which may translate into detection earlier in disease course. In this study, we sought to compare the sensitivity of RT-QuIC and IHC on a series of longitudinal tonsil and recto-anal mucosal lymphoid tissue (RAMALT) biopsies from white-tailed deer during the course of CWD progression.

**Methods:** White-tailed deer were inoculated via the per os (PO) route with CWD(+) [$n = 20$] and CWD(-) [$n = 4$] inocula (brain homogenate or saliva) and paired tonsil and RAMALT biopsies were collected every 3 months thereafter. Biopsies were assayed for prion seeding activity by RT-QuIC and for $PrP^{CWD}$ deposi-tion by IHC.

**Results:** RT-QuIC detected seeding activity in 10 of 20 (50%) tonsil biopsies prior to IHC detection, on average of 5.1 months (3-15 months) earlier. In RAMALT biopsies, detectable seeding activity was observed in 13 of 20 (65%) samples prior to IHC detection, on average 4.6 months (3-15 months) earlier. In the remaining biopsies, detection was concurrent by both methods. At no sampling point did a positive IHC result precede a positive RT-QuIC result. Of note, for biopsy samples that had already been determined posi-tive by both RT-QuIC and IHC in a previous collection, RT-QuIC detected seeding activity in seven (7) tonsil and four (4) RAMALT follow-up biopsies even though no lymphoid follicles were present in the paired sam-ples to assess positivity by IHC. All biopsies from CWD (-) deer were negative by both assays throughout the study. Overall, these results demonstrate that RT-QuIC detected CWD positivity in both tonsil and RAMALT biopsies approximately 5 months prior to IHC. Moreover, seeding activity was detectable even when

(due to poor biopsy sampling) lymphoid follicles were not present to permit meaningful IHC analysis.

**Conclusions:** These data indicate that RT-QuIC is more sensitive than IHC in detecting CWD infection, and could thereby be useful in determining future management strategies.

## Funding

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

## 99. Cellulose ethers interfere with CWD prion propagation in vitro and in vivo

Preetha Gopalakrishnanª, Jie Yuª, Samia Hannaouiª, Sheng Chun Changª, Maria Arifinª, Katsumi Doh-uraᵇ, Hermann Schatzlᶜ and Sabine Gilchª

ªDepartment of Ecosystem and Public Health, Calgary Prion Research Unit, Faculty of Veterinary Medicine; Hotchkiss Brain Institute; University of Calgary, Calgary, Canada; ᵇDepartment of Neurochemistry, Tohoku University Graduate School of Medicine, Sendai, Japan; ᶜDepartment of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine; Hotchkiss Brain Institute; University of Calgary, Canada

## ABSTRACT

Chronic wasting disease (CWD) is a prion disease of free ranging and farmed cervids. CWD is highly contagious and the absence of an effective vaccine makes a complete era-dication of CWD not realistic to date. Recently, a single subcutaneous injection of Cellulose Ethers (CEs) was reported to markedly extend the life span of transgenic mice infected with 263K prions. Despite the fact that the mechanisms of action of the CEs are still a matter of discussion, this approach could be a good strategy against CWD spreading, thus, limiting its potential in transmission to cervid and non-cervid animals and potentially to humans. In this study, we used the cellulose ethers TC-5RW and 60SH *in vitro* and *in vivo* to assess their efficacy to interfere with CWD prion propagation. *In vitro*, TC-5RW displays a dose dependant response to inhibit RT-QuIC reaction seeded with different CWD isolates from white-tailed deer (WTD), mule deer (MD) and elk. The inhibitory effect starts with a concentration as low as 0.1 µg/ml and totally inhibits conversion at 10 µg/ml. Groups of transgenic mice overexpressing elk $PrP^{C}$ were subcutaneously (s.c.) injected with the CE compounds and inoculated intracerebrally (i.c.) with different CWD isolates (elk, WTD, and MD) the same day (group A) or one month after the CE injection (group B). All treated groups show a significantly prolonged incubation period. Survival is extended between +12% to +32% in mice of group A, and +20% to +31% in mice of group B, depending on the
<figure><img src="image_65.png" /></figure>

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

CONTACT Nathaniel D. Denkers nathaniel.denkers@colostate.edu

*Present address: AstraZeneca, Waltham, NJ

## ABSTRACT

**Background:** Chronic wasting disease (CWD) continues to expand across North America and Canada, and more recently was discovered in Scandinavia. While the exact mechanism of CWD transmission has yet to be elucidated, early diagnosis of infected animals remains a priority in curtailing its spread. While immuno-histochemistry (IHC) and enzyme linked immunosorbent assay (ELISA) remain the gold standards for diagnosing CWD, real-time quaking induced conversion (RT-QuIC) is capable of detecting substantially lower concentrations of prions, which may translate into detection earlier in disease course. In this study, we sought to compare the sensitivity of RT-QuIC and IHC on a series of longitudinal tonsil and recto-anal mucosal lymphoid tissue (RAMALT) biopsies from white-tailed deer during the course of CWD progression.

**Methods:** White-tailed deer were inoculated via the per os (PO) route with CWD(+) [$n = 20$] and CWD(-) [$n = 4$] inocula (brain homogenate or saliva) and paired tonsil and RAMALT biopsies were collected every 3 months thereafter. Biopsies were assayed for prion seeding activity by RT-QuIC and for $PrP^{CWD}$ deposi-tion by IHC.

**Results:** RT-QuIC detected seeding activity in 10 of 20 (50%) tonsil biopsies prior to IHC detection, on average of 5.1 months (3-15 months) earlier. In RAMALT biopsies, detectable seeding activity was observed in 13 of 20 (65%) samples prior to IHC detection, on average 4.6 months (3-15 months) earlier. In the remaining biopsies, detection was concurrent by both methods. At no sampling point did a positive IHC result precede a positive RT-QuIC result. Of note, for biopsy samples that had already been determined posi-tive by both RT-QuIC and IHC in a previous collection, RT-QuIC detected seeding activity in seven (7) tonsil and four (4) RAMALT follow-up biopsies even though no lymphoid follicles were present in the paired sam-ples to assess positivity by IHC. All biopsies from CWD (-) deer were negative by both assays throughout the study. Overall, these results demonstrate that RT-QuIC detected CWD positivity in both tonsil and RAMALT biopsies approximately 5 months prior to IHC. Moreover, seeding activity was detectable even when

(due to poor biopsy sampling) lymphoid follicles were not present to permit meaningful IHC analysis.

**Conclusions:** These data indicate that RT-QuIC is more sensitive than IHC in detecting CWD infection, and could thereby be useful in determining future management strategies.

## Funding

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

## 99. Cellulose ethers interfere with CWD prion propagation in vitro and in vivo

Preetha Gopalakrishnanª, Jie Yuª, Samia Hannaouiª, Sheng Chun Changª, Maria Arifinª, Katsumi Doh-uraᵇ, Hermann Schatzlᶜ and Sabine Gilchª

ªDepartment of Ecosystem and Public Health, Calgary Prion Research Unit, Faculty of Veterinary Medicine; Hotchkiss Brain Institute; University of Calgary, Calgary, Canada; ᵇDepartment of Neurochemistry, Tohoku University Graduate School of Medicine, Sendai, Japan; ᶜDepartment of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine; Hotchkiss Brain Institute; University of Calgary, Canada

## ABSTRACT

Chronic wasting disease (CWD) is a prion disease of free ranging and farmed cervids. CWD is highly contagious and the absence of an effective vaccine makes a complete era-dication of CWD not realistic to date. Recently, a single subcutaneous injection of Cellulose Ethers (CEs) was reported to markedly extend the life span of transgenic mice infected with 263K prions. Despite the fact that the mechanisms of action of the CEs are still a matter of discussion, this approach could be a good strategy against CWD spreading, thus, limiting its potential in transmission to cervid and non-cervid animals and potentially to humans. In this study, we used the cellulose ethers TC-5RW and 60SH *in vitro* and *in vivo* to assess their efficacy to interfere with CWD prion propagation. *In vitro*, TC-5RW displays a dose dependant response to inhibit RT-QuIC reaction seeded with different CWD isolates from white-tailed deer (WTD), mule deer (MD) and elk. The inhibitory effect starts with a concentration as low as 0.1 µg/ml and totally inhibits conversion at 10 µg/ml. Groups of transgenic mice overexpressing elk $PrP^{C}$ were subcutaneously (s.c.) injected with the CE compounds and inoculated intracerebrally (i.c.) with different CWD isolates (elk, WTD, and MD) the same day (group A) or one month after the CE injection (group B). All treated groups show a significantly prolonged incubation period. Survival is extended between +12% to +32% in mice of group A, and +20% to +31% in mice of group B, depending on the
<figure><img src="image_62.png" /></figure>

CWD isolate used for inoculation, when compared to the control group. The prolonged incubation period in the treated groups correlates to a significantly reduced resistance of prions to proteinase K (PK). This is demonstrated by Western blot analyses of 10% brain homogenate digested with a range of PK concentration. Interestingly, upon passage of brain homogenates from control and group B mice, tgElk mice inoculated with group B material showed a prolonged life span of +17% without further CE treatment, supporting the hypothesis of a modified state of infectivity in the mice treated during first passage. In summary, our results demonstrate that CEs are efficient in inhibiting CWD prion propagation independent of the origin of the isolates used for inoculation. We show that CE treatment alters biochemical properties and reduces infectivity of prions *in vivo*. Therefore, these compounds can be useful in limiting the spread of CWD.

## 100. RT-QuIC detection of pathological prion protein in subclinical goats following experimental oral transmission of L-type BSE

Alessandra Favole<sup>a</sup>, Maria Mazza<sup>a</sup>, Elena Vallino Costassa<sup>a</sup>, Antonio D'Angelo<sup>b</sup>, Guerino Lombardi<sup>c</sup>, Claudia Palmitessa<sup>a</sup>, Dell'Atti Luana<sup>a</sup>, Paola Crociara<sup>a</sup>, Elena Berrone<sup>a</sup>, Marina Gallo<sup>a</sup>, Monica Lo Faro<sup>a</sup>, Tiziana Avanzato<sup>a</sup>, Pier Luigi Acutis<sup>a</sup>, Cristina Casalone<sup>a</sup> and Cristiano Corona<sup>a</sup>

CONTACT Alessandra Favole alessandra.favole@izsto.it

### ABSTRACT

In vitro assays such as serial protein misfolding amplification and real-time quaking-induced conversion (RT-QuIC) allow assessment of the conversion of the cellular prion protein (PrP<sup>C</sup>) into a pathogenic misfolded form (PrP<sup>Sc</sup>). RT-QuIC can be used for the detection of typical and atypical prions in a variety of biological tissues from humans and animals. It has been demonstrated that L-type Bovine Spongiform Encephalopathy (L-BSE) can be transmitted to a range of species including sheep<sup>1</sup> and goats<sup>2</sup>, by intracranial inoculation. However, information on susceptibility of small ruminants to atypical BSE prions through oral transmission are still lacking.

In the present work four Saanen and one mixed breed goats were orally administered with 15 ml of a 30% (wt/vol) L-type BSE brain tissue homogenate (5 g tissue). Despite a prolonged observation period of 80 months, all the animals did not develop clinical signs referable to TSEs and tested

negative by standard diagnostics performed on brainstem. Next, pooled brain samples from the same animals were analyzed by western blot (WB) and RT-QuIC by using hamster-sheep (Ha-S) and Ha 90-231 recombinant PrP<sup>Sen</sup>. All the examined samples resulted negative at WB whereas three out of five showed positive RT-QuIC reactions. Furthermore, prion distribution in multiple brain regions was assessed in two out of three goats resulted positive. Preliminary results demonstrated RT-QuIC seedling activity in various brain regions including frontal cortex and cerebellum. Our data confirm the susceptibility of goat species to bovine TSEs and the applicability of RT-QuIC as a highly sensitive assay able to detect prions in subclinical animals. Furthermore, based on the results achieved in this study, a revision or implementation of traditional methods of diagnosis could be obtained.

### References

[1] Simmons MM, et al. Vet Res. 2016;47(1):112.

[2] Vallino Costassa E, et al. PLoS One. 2018;13(5):e0198037.

## 101. Application of high-throughput, capillary-based Western immunoassays to modulated cleavage of PrP<sup>C</sup>

Andrew R. Castle<sup>a,b</sup>, Nathalie Daude<sup>a</sup>, Sabine Gilch<sup>c</sup> and David Westaway<sup>a,b,d</sup>

CONTACT Andrew R. Castle acastle@ualberta.ca

### ABSTRACT

The C-terminal fragment of the cellular prion protein (PrP<sup>C</sup>) that is generated physiologically by the alpha-cleavage pathway is resistant to misfolding and acts as a dominant-negative inhibitor of prion replication. This fragment, known as C1, also lacks the reported binding sites for neurotoxic beta-amyloid and alpha-synuclein oligomers that are present in full length (FL) PrP<sup>C</sup>. In contrast, a rarer proteolytic pathway – beta-cleavage – produces the longer C-terminal fragment C2 that retains the ability to misfold and may be resistant to cleavage at the ‘alpha’ site. Thus, modulators of PrP<sup>C</sup> proteolysis that enhance alpha-cleavage or inhibit beta-cleavage are of interest therapeutically. To identify such modulators, we initiated cell-based screens of small molecule libraries to identify compounds with

<footnote><sup>a</sup>Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'Aosta; <sup>b</sup>University of Turin, Department of Veterinary Science; <sup>c</sup>Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna</footnote>

<footnote><sup>a</sup>Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Canada; <sup>b</sup>Department of Medicine, University of Alberta, Edmonton, Canada; <sup>c</sup>Department of Ecosystem and Public Health, Calgary Prion Research Unit, Faculty of Veterinary Medicine and Hotchkiss Brain Institute, University of Calgary, Calgary, Canada; <sup>d</sup>Department of Biochemistry, University of Alberta, Edmonton, Canada</footnote>
<figure><img src="image_66.png" /></figure>

CWD isolate used for inoculation, when compared to the control group. The prolonged incubation period in the treated groups correlates to a significantly reduced resistance of prions to proteinase K (PK). This is demonstrated by Western blot analyses of 10% brain homogenate digested with a range of PK concentration. Interestingly, upon passage of brain homogenates from control and group B mice, tgElk mice inoculated with group B material showed a prolonged life span of +17% without further CE treatment, supporting the hypothesis of a modified state of infectivity in the mice treated during first passage. In summary, our results demonstrate that CEs are efficient in inhibiting CWD prion propagation independent of the origin of the isolates used for inoculation. We show that CE treatment alters biochemical properties and reduces infectivity of prions *in vivo*. Therefore, these compounds can be useful in limiting the spread of CWD.

## 100. RT-QuIC detection of pathological prion protein in subclinical goats following experimental oral transmission of L-type BSE

Alessandra Favole<sup>a</sup>, Maria Mazza<sup>a</sup>, Elena Vallino Costassa<sup>a</sup>, Antonio D'Angelo<sup>b</sup>, Guerino Lombardi<sup>c</sup>, Claudia Palmitessa<sup>a</sup>, Dell'Atti Luana<sup>a</sup>, Paola Crociara<sup>a</sup>, Elena Berrone<sup>a</sup>, Marina Gallo<sup>a</sup>, Monica Lo Faro<sup>a</sup>, Tiziana Avanzato<sup>a</sup>, Pier Luigi Acutis<sup>a</sup>, Cristina Casalone<sup>a</sup> and Cristiano Corona<sup>a</sup>

CONTACT Alessandra Favole alessandra.favole@izsto.it

### ABSTRACT

In vitro assays such as serial protein misfolding amplification and real-time quaking-induced conversion (RT-QuIC) allow assessment of the conversion of the cellular prion protein (PrP<sup>C</sup>) into a pathogenic misfolded form (PrP<sup>Sc</sup>). RT-QuIC can be used for the detection of typical and atypical prions in a variety of biological tissues from humans and animals. It has been demonstrated that L-type Bovine Spongiform Encephalopathy (L-BSE) can be transmitted to a range of species including sheep<sup>1</sup> and goats<sup>2</sup>, by intracranial inoculation. However, information on susceptibility of small ruminants to atypical BSE prions through oral transmission are still lacking.

In the present work four Saanen and one mixed breed goats were orally administered with 15 ml of a 30% (wt/vol) L-type BSE brain tissue homogenate (5 g tissue). Despite a prolonged observation period of 80 months, all the animals did not develop clinical signs referable to TSEs and tested

negative by standard diagnostics performed on brainstem. Next, pooled brain samples from the same animals were analyzed by western blot (WB) and RT-QuIC by using hamster-sheep (Ha-S) and Ha 90-231 recombinant PrP<sup>Sen</sup>. All the examined samples resulted negative at WB whereas three out of five showed positive RT-QuIC reactions. Furthermore, prion distribution in multiple brain regions was assessed in two out of three goats resulted positive. Preliminary results demonstrated RT-QuIC seedling activity in various brain regions including frontal cortex and cerebellum. Our data confirm the susceptibility of goat species to bovine TSEs and the applicability of RT-QuIC as a highly sensitive assay able to detect prions in subclinical animals. Furthermore, based on the results achieved in this study, a revision or implementation of traditional methods of diagnosis could be obtained.

### References

[1] Simmons MM, et al. Vet Res. 2016;47(1):112.

[2] Vallino Costassa E, et al. PLoS One. 2018;13(5):e0198037.

## 101. Application of high-throughput, capillary-based Western immunoassays to modulated cleavage of PrP<sup>C</sup>

Andrew R. Castle<sup>a,b</sup>, Nathalie Daude<sup>a</sup>, Sabine Gilch<sup>c</sup> and David Westaway<sup>a,b,d</sup>

CONTACT Andrew R. Castle acastle@ualberta.ca

### ABSTRACT

The C-terminal fragment of the cellular prion protein (PrP<sup>C</sup>) that is generated physiologically by the alpha-cleavage pathway is resistant to misfolding and acts as a dominant-negative inhibitor of prion replication. This fragment, known as C1, also lacks the reported binding sites for neurotoxic beta-amyloid and alpha-synuclein oligomers that are present in full length (FL) PrP<sup>C</sup>. In contrast, a rarer proteolytic pathway – beta-cleavage – produces the longer C-terminal fragment C2 that retains the ability to misfold and may be resistant to cleavage at the ‘alpha’ site. Thus, modulators of PrP<sup>C</sup> proteolysis that enhance alpha-cleavage or inhibit beta-cleavage are of interest therapeutically. To identify such modulators, we initiated cell-based screens of small molecule libraries to identify compounds with

<footnote><sup>a</sup>Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'Aosta; <sup>b</sup>University of Turin, Department of Veterinary Science; <sup>c</sup>Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna</footnote>

<footnote><sup>a</sup>Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Canada; <sup>b</sup>Department of Medicine, University of Alberta, Edmonton, Canada; <sup>c</sup>Department of Ecosystem and Public Health, Calgary Prion Research Unit, Faculty of Veterinary Medicine and Hotchkiss Brain Institute, University of Calgary, Calgary, Canada; <sup>d</sup>Department of Biochemistry, University of Alberta, Edmonton, Canada</footnote>
<figure><img src="image_63.png" /></figure>

effects on PrP<sup>C</sup> proteolysis. We decided to measure the levels of FL PrP<sup>C</sup> and its cleavage fragments using an automated, capillary-based Western immunoassay system; this technique offers higher throughput and more accurate quantification than traditional Western blotting. Firstly, we optimised the detection and quantification of FL PrP<sup>C</sup> and its cleavage fragments in cell lysate samples treated with peptide-N-glycosidase F to remove the N-linked glycans normally present. Secondly, we optimised the simultaneous quantification of PrP<sup>C</sup> and beta-tubulin levels to allow for loading error correction. Thirdly, we developed a protocol for performing all cell culture, lysis and deglycosylation steps in 96-well microplates before capillary Western analysis and demonstrated that this approach resulted in comparable intra-assay percentage coefficients of variation to using standard protocols for these steps. Finally, we screened the ApexBio DiscoveryProbe Protease Inhibitor Library by exposing RK13 cells expressing the artificial 'S3' variant of PrP<sup>C</sup> to each compound at 20 µM concentration for 4 days; S3 PrP<sup>C</sup> displays accentuated beta-cleavage in RK13 cells, thereby enabling C2 levels to be quantified reliably. Analysis of the screening data revealed distinct classes of inhibitors with differing effects on PrP<sup>C</sup> fragmentation products.

**KEYWORDS:** Prion disease; neurodegeneration; protein misfolding; proteolysis; fragmentation; high-throughput screening; capillary Western

**102. Distinctive PrP particles comprise hamster-adapted prion strains**

Leonardo M. Cortez<sup>a,b</sup>, Camilo Duque Velázquez<sup>a,b</sup>, Debbie McKenzie<sup>a,c</sup>, Valerie L. Sim<sup>a,b</sup>

CONTACT Leonardo M. Cortez [lcortez@ualberta.ca](mailto:lcortez@ualberta.ca)

**ABSTRACT**

**Background:** The conversion of PrP<sup>C</sup> into a pathogenic conformation results in multimeric PrP<sup>Sc</sup> particles that range in size from small detergent-soluble oligomers to large insoluble aggregates. The prion strain phenomenon, characterized by different phenotypes/pathologies of disease, may be explained by PrP<sup>Sc</sup> conformational differences resulting in reproducible strain-specific PrP<sup>Sc</sup> multimeric states. However, the complexity, variability, and properties of these PrP<sup>Sc</sup> particle distributions are not well established. It has been hypothesized that prion strains are comprised of a cloud of PrP<sup>Sc</sup> conformations with different aggregation properties. We present a detailed study of fractionated

PrP<sup>Sc</sup> particles to better understand prion strains, transmission barriers, drug resistance, and therapeutic targets.

**Methods:** Asymmetric-flow field-flow fractionation (AF4), a one-phase chromatography technique of size-based separation, was used to isolate soluble prion particles from brain homogenate of 263K, Hyper (HY) and Drowsy (DY) infected hamsters. We determined the hydrodynamic radius (Rh) and amount of total and PK-resistant PrP in each fraction using dynamic light scattering and immunoblotting. Using different AF4 buffer conditions, we studied the stability of different PrP oligomers. Particle size was correlated with RT-QuIC seeding activity.

**Results:** Each strain generated a reproducible distribution of PrP particle sizes. For HY and 263K, there was a population of smaller PrP particles (Rh = 2-8nm) that were sensitive to PK digestion but had strong seeding activity. A second population of larger PrP particles (Rh~15-200nm) was detergent and PK resistant but had less seeding activity than the smaller particles. In contrast, DY had predominantly a smaller population of PrP particles, peaking at Rh~7nm. These smaller DY particles were sensitive to PK but had less seeding activity than 263K and HY. The few large DY PrP particles were resistant to PK but sensitive to detergent, and their seeding activity was stronger than large HY and 263K PrP particles.

Interestingly, the size distribution of PK-resistant PrP particles was almost identical for all strains, with PK resistance being evident in particles with Rh>20nm. This suggests that PK resistance is related to the size of PrP particle.

**Conclusions:** The distinct PrP<sup>Sc</sup> particle size distribution and oligomer properties in different strains suggest there are mechanisms to maintain a stable PrP<sup>Sc</sup> population in each strain. Hyper and 263K, which have similar disease outcomes, are comprised of similar distributions of PrP<sup>Sc</sup> particles; Drowsy, with very a different disease outcome, has different amounts of aggregates with different biochemical properties. PK sensitive PrP appears to play a central role in these differences.

**103. Biochemical comparison of peripheral vs. central nervous system-derived PrP<sup>CWD</sup> in white-tailed deer**

Lindsay E. Parrie, Davin M. Henderson, Clare Hoover, Kristen A. Davenport, Sarah K. Cooper and Edward A. Hoover

Prion Research Center, Colorado State University, Fort Collins, CO, USA

CONTACT Lindsay E. Parrie [lindsay.parrie@colostate.edu](mailto:lindsay.parrie@colostate.edu)

**ABSTRACT**

In nature, cervids are infected with chronic wasting disease (CWD) by oral and nasal mucosal exposure. However, PrP<sup>C</sup> expression is not the primary driver of PrP<sup>CWD</sup>

<footnote><sup>a</sup>Centre for Prions and Protein Folding Diseases, University of Alberta; <sup>b</sup>Department of Medicine, Faculty of Medicine & Dentistry, University of Alberta; <sup>c</sup>Department of Biological Sciences, Faculty of Science, University of Alberta</footnote>
<figure><img src="image_67.png" /></figure>

effects on PrP<sup>C</sup> proteolysis. We decided to measure the levels of FL PrP<sup>C</sup> and its cleavage fragments using an automated, capillary-based Western immunoassay system; this technique offers higher throughput and more accurate quantification than traditional Western blotting. Firstly, we optimised the detection and quantification of FL PrP<sup>C</sup> and its cleavage fragments in cell lysate samples treated with peptide-N-glycosidase F to remove the N-linked glycans normally present. Secondly, we optimised the simultaneous quantification of PrP<sup>C</sup> and beta-tubulin levels to allow for loading error correction. Thirdly, we developed a protocol for performing all cell culture, lysis and deglycosylation steps in 96-well microplates before capillary Western analysis and demonstrated that this approach resulted in comparable intra-assay percentage coefficients of variation to using standard protocols for these steps. Finally, we screened the ApexBio DiscoveryProbe Protease Inhibitor Library by exposing RK13 cells expressing the artificial 'S3' variant of PrP<sup>C</sup> to each compound at 20 µM concentration for 4 days; S3 PrP<sup>C</sup> displays accentuated beta-cleavage in RK13 cells, thereby enabling C2 levels to be quantified reliably. Analysis of the screening data revealed distinct classes of inhibitors with differing effects on PrP<sup>C</sup> fragmentation products.

**KEYWORDS:** Prion disease; neurodegeneration; protein misfolding; proteolysis; fragmentation; high-throughput screening; capillary Western

**102. Distinctive PrP particles comprise hamster-adapted prion strains**

Leonardo M. Cortez<sup>a,b</sup>, Camilo Duque Velázquez<sup>a,b</sup>, Debbie McKenzie<sup>a,c</sup>, Valerie L. Sim<sup>a,b</sup>

CONTACT Leonardo M. Cortez lcortez@ualberta.ca

**ABSTRACT**

**Background:** The conversion of PrP<sup>C</sup> into a pathogenic conformation results in multimeric PrP<sup>Sc</sup> particles that range in size from small detergent-soluble oligomers to large insoluble aggregates. The prion strain phenomenon, characterized by different phenotypes/pathologies of disease, may be explained by PrP<sup>Sc</sup> conformational differences resulting in reproducible strain-specific PrP<sup>Sc</sup> multimeric states. However, the complexity, variability, and properties of these PrP<sup>Sc</sup> particle distributions are not well established. It has been hypothesized that prion strains are comprised of a cloud of PrP<sup>Sc</sup> conformations with different aggregation properties. We present a detailed study of fractionated

PrP<sup>Sc</sup> particles to better understand prion strains, transmission barriers, drug resistance, and therapeutic targets.

**Methods:** Asymmetric-flow field-flow fractionation (AF4), a one-phase chromatography technique of size-based separation, was used to isolate soluble prion particles from brain homogenate of 263K, Hyper (HY) and Drowsy (DY) infected hamsters. We determined the hydrodynamic radius (Rh) and amount of total and PK-resistant PrP in each fraction using dynamic light scattering and immunoblotting. Using different AF4 buffer conditions, we studied the stability of different PrP oligomers. Particle size was correlated with RT-QuIC seeding activity.

**Results:** Each strain generated a reproducible distribution of PrP particle sizes. For HY and 263K, there was a population of smaller PrP particles (Rh = 2-8nm) that were sensitive to PK digestion but had strong seeding activity. A second population of larger PrP particles (Rh~15-200nm) was detergent and PK resistant but had less seeding activity than the smaller particles. In contrast, DY had predominantly a smaller population of PrP particles, peaking at Rh~7nm. These smaller DY particles were sensitive to PK but had less seeding activity than 263K and HY. The few large DY PrP particles were resistant to PK but sensitive to detergent, and their seeding activity was stronger than large HY and 263K PrP particles.

Interestingly, the size distribution of PK-resistant PrP particles was almost identical for all strains, with PK resistance being evident in particles with Rh>20nm. This suggests that PK resistance is related to the size of PrP particle.

**Conclusions:** The distinct PrP<sup>Sc</sup> particle size distribution and oligomer properties in different strains suggest there are mechanisms to maintain a stable PrP<sup>Sc</sup> population in each strain. Hyper and 263K, which have similar disease outcomes, are comprised of similar distributions of PrP<sup>Sc</sup> particles; Drowsy, with very a different disease outcome, has different amounts of aggregates with different biochemical properties. PK sensitive PrP appears to play a central role in these differences.

**103. Biochemical comparison of peripheral vs. central nervous system-derived PrP<sup>CWD</sup> in white-tailed deer**

Lindsay E. Parrie, Davin M. Henderson, Clare Hoover, Kristen A. Davenport, Sarah K. Cooper and Edward A. Hoover

Prion Research Center, Colorado State University, Fort Collins, CO, USA

CONTACT Lindsay E. Parrie lindsay.parrie@colostate.edu

**ABSTRACT**

In nature, cervids are infected with chronic wasting disease (CWD) by oral and nasal mucosal exposure. However, PrP<sup>C</sup> expression is not the primary driver of PrP<sup>CWD</sup>

<footnote><sup>a</sup>Centre for Prions and Protein Folding Diseases, University of Alberta; <sup>b</sup>Department of Medicine, Faculty of Medicine & Dentistry, University of Alberta; <sup>c</sup>Department of Biological Sciences, Faculty of Science, University of Alberta</footnote>
<figure><img src="image_64.png" /></figure>

accumulation, as tissues with the highest $PrP^C$ expression are not the first to accumulate $PrP^{CWD}$. Studies of early CWD pathogenesis have implicated pharyngeal lymphoid tissues as the earliest sites of prion accumulation. $PrP^{CWD}$ replication differs between peripheral tissues and the CNS. $PrP^{CWD}$ deposits in lymphoid tissues are smaller and more diffuse than those found in brain, which is typically characterized by large amyloid plaques. Additionally, lymphoid-derived $PrP^{CWD}$ appears to be more sensitive to proteinase K digestion than is brain $PrP^{CWD}$, despite equivalent seeding activity as measured in RT-QuIC. Our studies address two main questions: (1) Do lymphoid prions have different biophysical properties than those from the CNS? (2) Do lymphoid prions have different infectivity properties than CNS prions? To investigate the potential differences between peripheral $PrP^{CWD}$ and prions formed in the CNS, we orally exposed white-tailed deer (WTD) to very low doses of infectious CWD+ saliva or brain homogenate. Tissues were collected at clinical disease, and prions from retropharyngeal lymph node and obex compared as to biophysical properties, conversion seeding efficiency, protease sensitivity, and immuno-blotting profiles. To functionally assess infectivity of peripheral and CNS-derived $PrP^{CWD}$, we will perform both cervid prion cell assay (CPCA) and bioassay of cervidized transgenic mice via oral inoculation. These studies will illuminate previously unknown features of CWD peripheral prion pathogenesis that likely relate to the efficient horizontal transmission of CWD.

**KEYWORDS:** Chronic wasting disease; lymph node; RT-QuIC

# 104. Development of a high-throughput assay for tau purification and aggregation

Allan Yarahmady and Sue-Ann Mok

Department of Biochemistry, University of Alberta, Edmonton, Canada

**CONTACT** Allan Yarahmady yarahmad@ualberta.ca

## ABSTRACT

**Introduction:** To study the process of tau aggregation, researchers have commonly utilized *in vitro* experiments where tau expressed and purified from *E. coli* is aggregated via addition of inducers such as heparin. However, the structures of artificially obtained tau fibrils differ from those found in actual Alzheimer's disease patients. Differences in structures suggest that the folding pathways of *in vitro* aggregation differ from those *in vivo*. These differences call into question the relevance of *in vitro* aggregation models. Development of potential treatments

that inhibit or reverse tau aggregation would likely be aided by *in vitro* derived fibrils that more closely mimic the structures observed in Alzheimer's patients. Point-mutations of tau have been shown to significantly alter the core structure of the generated fibrils. New methodologies such as deep mutational scanning of the tau sequence could identify specific mutants that give a fibril core structure identical to that of patient-derived fibrils. In order to perform a large-scale screen of tau mutants, methods for high-throughput expression, purification, and aggregate analysis of tau were optimized.

**Materials and Methods:** Tau was expressed in three millilitre cultures in 48-well deep plates. The pellets were lysed with lysozyme and boiled to denature contaminant proteins and cell debris. The lysates were spun down and the supernatants were isolated which contain soluble tau. The kinetics of tau aggregation were studied via a thioflavin T time-course assay using heparin as an inducer. Core structures of the aggregates from the kinetic assays were characterized using trypsin digestion and visualization by SDS-PAGE.

**Results:** It was found that tau was readily expressed and purified in small-scale. The yield from high-throughput small-scale expression of tau is consistent and the aggregation propensity of tau is able to be studied using a low-volume thioflavin T kinetic assay. The trypsin-resistant core of the fibrils derived from this assay resembles those obtained through traditional purification and *in vitro* aggregation techniques.

**Conclusions:** It is possible to obtain recombinantly expressed tau of high yield and purity using small-scale methods. The aggregation kinetics and fibril core structures of these tau extracts are also able to be characterized by thioflavin T assay and trypsin digestion, respectively. As such, a high-throughput deep mutational screen of tau is possible and can be used to test if the tau fibril structures observed in Alzheimer's can be recapitulated *in vitro*.

# 105. Differential binding of prions to vegetation

Elizabeth Triscott<sup>a</sup>, Alsu Kuznetsova<sup>b</sup>, Debbie McKenzie<sup>a</sup> and Judd Aiken<sup>c</sup>

**CONTACT** Elizabeth Triscott etriscot@ualberta.ca

## ABSTRACT

Chronic wasting disease (CWD), a prion disease of cervids, is present in North America and Europe. It affects a variety of captive and free ranging animals,

<footnote><sup>a</sup>Centre for Prions and Protein Folding Diseases, Department of Biological Sciences, University of Alberta; <sup>b</sup>Department of Renewable Resources, University of Alberta; <sup>c</sup>Centre for Prions and Protein Folding Diseases, Department of Agriculture Food and Nutritional Sciences, University of Alberta</footnote>
<figure><img src="image_68.png" /></figure>

accumulation, as tissues with the highest $PrP^C$ expression are not the first to accumulate $PrP^{CWD}$. Studies of early CWD pathogenesis have implicated pharyngeal lymphoid tissues as the earliest sites of prion accumulation. $PrP^{CWD}$ replication differs between peripheral tissues and the CNS. $PrP^{CWD}$ deposits in lymphoid tissues are smaller and more diffuse than those found in brain, which is typically characterized by large amyloid plaques. Additionally, lymphoid-derived $PrP^{CWD}$ appears to be more sensitive to proteinase K digestion than is brain $PrP^{CWD}$, despite equivalent seeding activity as measured in RT-QuIC. Our studies address two main questions: (1) Do lymphoid prions have different biophysical properties than those from the CNS? (2) Do lymphoid prions have different infectivity properties than CNS prions? To investigate the potential differences between peripheral $PrP^{CWD}$ and prions formed in the CNS, we orally exposed white-tailed deer (WTD) to very low doses of infectious CWD+ saliva or brain homogenate. Tissues were collected at clinical disease, and prions from retropharyngeal lymph node and obex compared as to biophysical properties, conversion seeding efficiency, protease sensitivity, and immuno-blotting profiles. To functionally assess infectivity of peripheral and CNS-derived $PrP^{CWD}$, we will perform both cervid prion cell assay (CPCA) and bioassay of cervidized transgenic mice via oral inoculation. These studies will illuminate previously unknown features of CWD peripheral prion pathogenesis that likely relate to the efficient horizontal transmission of CWD.

**KEYWORDS:** Chronic wasting disease; lymph node; RT-QuIC

# 104. Development of a high-throughput assay for tau purification and aggregation

Allan Yarahmady and Sue-Ann Mok

Department of Biochemistry, University of Alberta, Edmonton, Canada

**CONTACT** Allan Yarahmady yarahmad@ualberta.ca

## ABSTRACT

**Introduction:** To study the process of tau aggregation, researchers have commonly utilized *in vitro* experiments where tau expressed and purified from *E. coli* is aggregated via addition of inducers such as heparin. However, the structures of artificially obtained tau fibrils differ from those found in actual Alzheimer's disease patients. Differences in structures suggest that the folding pathways of *in vitro* aggregation differ from those *in vivo*. These differences call into question the relevance of *in vitro* aggregation models. Development of potential treatments

that inhibit or reverse tau aggregation would likely be aided by *in vitro* derived fibrils that more closely mimic the structures observed in Alzheimer's patients. Point-mutations of tau have been shown to significantly alter the core structure of the generated fibrils. New methodologies such as deep mutational scanning of the tau sequence could identify specific mutants that give a fibril core structure identical to that of patient-derived fibrils. In order to perform a large-scale screen of tau mutants, methods for high-throughput expression, purification, and aggregate analysis of tau were optimized.

**Materials and Methods:** Tau was expressed in three millilitre cultures in 48-well deep plates. The pellets were lysed with lysozyme and boiled to denature contaminant proteins and cell debris. The lysates were spun down and the supernatants were isolated which contain soluble tau. The kinetics of tau aggregation were studied via a thioflavin T time-course assay using heparin as an inducer. Core structures of the aggregates from the kinetic assays were characterized using trypsin digestion and visualization by SDS-PAGE.

**Results:** It was found that tau was readily expressed and purified in small-scale. The yield from high-throughput small-scale expression of tau is consistent and the aggregation propensity of tau is able to be studied using a low-volume thioflavin T kinetic assay. The trypsin-resistant core of the fibrils derived from this assay resembles those obtained through traditional purification and *in vitro* aggregation techniques.

**Conclusions:** It is possible to obtain recombinantly expressed tau of high yield and purity using small-scale methods. The aggregation kinetics and fibril core structures of these tau extracts are also able to be characterized by thioflavin T assay and trypsin digestion, respectively. As such, a high-throughput deep mutational screen of tau is possible and can be used to test if the tau fibril structures observed in Alzheimer's can be recapitulated *in vitro*.

# 105. Differential binding of prions to vegetation

Elizabeth Triscott<sup>a</sup>, Alsu Kuznetsova<sup>b</sup>, Debbie McKenzie<sup>a</sup> and Judd Aiken<sup>c</sup>

**CONTACT** Elizabeth Triscott etriscot@ualberta.ca

## ABSTRACT

Chronic wasting disease (CWD), a prion disease of cervids, is present in North America and Europe. It affects a variety of captive and free ranging animals,

<footnote><sup>a</sup>Centre for Prions and Protein Folding Diseases, Department of Biological Sciences, University of Alberta; <sup>b</sup>Department of Renewable Resources, University of Alberta; <sup>c</sup>Centre for Prions and Protein Folding Diseases, Department of Agriculture Food and Nutritional Sciences, University of Alberta</footnote>
<figure><img src="image_65.png" /></figure>

including mule deer, white tailed deer, reindeer, moose, and elk. Preclinical and clinical animals shed infectivity in a number of bodily fluids, including saliva, faeces, and nasal secretions. These prions are resistant to degradation and can contaminate the environment for many years. Factors such as the surface composition of materials and the mineralogy of soils can affect where infectivity accumulates in the environment. Given, however, that in most environments prions will initially contact vegetation, understanding the factors that affect the interaction between prion infectivity and vegetation is of utmost importance. We developed an assay in which infectious brain homogenate is applied to vegetation samples, which are then rinsed. The proteins are precipitated from the rinse water and the amount of PrP that rinses off each vegetation sample was compared. Given that surface composition affects prion adherence, we first compared lichen, which is coated with polysaccharides, and grass, which is covered with a waxy cuticle. Our data indicate that less prions rinse off lichen than grass. Furthermore, the binding of CWD prions to a variety of plants shows that the prion-vegetation interaction is affected by properties of the plant, e.g. microarchitecture and precise chemical composition. Freezing and drying of the plants alters this interaction. These data give us insight in how infectivity moves through the environment and has ramifications for the plausibility of environmental CWD transmission. Understanding where prions accumulate will help in developing methods for environmental monitoring and decontamination.

**106. Long-read nanopore sequencing provides fast and accurate identification of genetic variants in the human PRNP gene**

Dimitriadis Athanasios, Francois Kroll, Tracy Campbell, John Collinge, Simon Mead, Emmanuelle Viré and John Collinge

MRC Prion Unit at UCL, Institute of Prion Diseases, Queen Square House, Queen Square, University College London, London, UK

CONTACT Dimitriadis Athanasios athanasios.dimitriadis.16@ucl.ac.uk

**ABSTRACT**

The human prion gene (PRNP) is a 15.5 kb sequence on chromosome 20 which encodes the prion protein (PrP). It is composed of two exons flanking a single large intron. The protein coding region is a 762-bp region within the second exon. Genetic variants identified in the PRNP gene are associated with prion diseases, such as Creutzfeldt-Jakob disease (CJD). Previous work has demonstrated

that the number of OctaPeptide Repeats (OPRs) in the gene coding region is in general inversely correlated with age of disease onset [1]. Similarly, it is well established that genotype of the codon 129 strongly affects disease onset, severity, and phenotype [2]. This evidence highlights the necessity of accurate genetic testing in CJD management. Here we tested how long-read sequencing technologies compare to the well-established Sanger sequencing method, using a Nanopore MinION sequencer. First, we sequenced full-length PRNP in 9 individuals known to be carrying various types of OPR insertions (OPRIs), deletions (OPRDs), and SNVs (E200K, P102L) and confirmed their genotype. To process this data, we designed a bioinformatics pipeline based on minimap2, sniffles, nanopolish, and custom R scripts to automate the analysis and filter our results. Then, we sequenced the full-length prion gene in 10 patients with sporadic CJD and characterized polymorphisms in the intronic region and the promoter of the gene. Altogether our results reveal that long-read sequencing, using MinION, allows investigating the genetic makeup of the whole PRNP in patients, including the promoter, flanking regions, intron, and both exons of the gene. We propose an accurate, quick, and user friendly method that bypasses the needs for expensive instruments and which allows for sample multiplexing. Finally, our pipeline is also able to report the results in an easily readable text format.

**KEYWORDS:**

Nanopore sequencing; genetic prion disease; bioinformatics; structural variants; single-nucleotide variations

**References**

[1] Brown K, et al. J Geriatr Psychiatry Neurol. 2010;23(4):277-298.

[2] Schmitz M, et al. Mol Neurobiol. 2017;54(6):4138-4149.

**107. Global analysis of protein degradation rates in prion infected cells by dynamic SILAC**

Charles R. Hutti, Kevin A. Welle, Jennifer R. Hryhorenko and Sina Ghaemmaghami

University of Rochester, New York

CONTACT Charles R. Hutti m.shafiq@uke.de

**ABSTRACT**

Prions diseases are rare neurological disorders caused by the misfolding of cellular prion protein (PrPC). The misfolded conformers aggregate into cytotoxic fibrils (PrPSc) that facilitate the conversion of additional prion proteins into the misfolded form. Intracellular PrPSc aggregates primarily
<figure><img src="image_69.png" /></figure>

including mule deer, white tailed deer, reindeer, moose, and elk. Preclinical and clinical animals shed infectivity in a number of bodily fluids, including saliva, faeces, and nasal secretions. These prions are resistant to degradation and can contaminate the environment for many years. Factors such as the surface composition of materials and the mineralogy of soils can affect where infectivity accumulates in the environment. Given, however, that in most environments prions will initially contact vegetation, understanding the factors that affect the interaction between prion infectivity and vegetation is of utmost importance. We developed an assay in which infectious brain homogenate is applied to vegetation samples, which are then rinsed. The proteins are precipitated from the rinse water and the amount of PrP that rinses off each vegetation sample was compared. Given that surface composition affects prion adherence, we first compared lichen, which is coated with polysaccharides, and grass, which is covered with a waxy cuticle. Our data indicate that less prions rinse off lichen than grass. Furthermore, the binding of CWD prions to a variety of plants shows that the prion-vegetation interaction is affected by properties of the plant, e.g. microarchitecture and precise chemical composition. Freezing and drying of the plants alters this interaction. These data give us insight in how infectivity moves through the environment and has ramifications for the plausibility of environmental CWD transmission. Understanding where prions accumulate will help in developing methods for environmental monitoring and decontamination.

**106. Long-read nanopore sequencing provides fast and accurate identification of genetic variants in the human PRNP gene**

Dimitriadis Athanasios, Francois Kroll, Tracy Campbell, John Collinge, Simon Mead, Emmanuelle Viré and John Collinge

MRC Prion Unit at UCL, Institute of Prion Diseases, Queen Square House, Queen Square, University College London, London, UK

CONTACT Dimitriadis Athanasios athanasios.dimitriadis.16@ucl.ac.uk

ABSTRACT

The human prion gene (PRNP) is a 15.5 kb sequence on chromosome 20 which encodes the prion protein (PrP). It is composed of two exons flanking a single large intron. The protein coding region is a 762-bp region within the second exon. Genetic variants identified in the PRNP gene are associated with prion diseases, such as Creutzfeldt-Jakob disease (CJD). Previous work has demonstrated

that the number of OctaPeptide Repeats (OPRs) in the gene coding region is in general inversely correlated with age of disease onset [1]. Similarly, it is well established that genotype of the codon 129 strongly affects disease onset, severity, and phenotype [2]. This evidence highlights the necessity of accurate genetic testing in CJD management. Here we tested how long-read sequencing technologies compare to the well-established Sanger sequencing method, using a Nanopore MinION sequencer. First, we sequenced full-length PRNP in 9 individuals known to be carrying various types of OPR insertions (OPRIs), deletions (OPRDs), and SNVs (E200K, P102L) and confirmed their genotype. To process this data, we designed a bioinformatics pipeline based on minimap2, sniffles, nanopolish, and custom R scripts to automate the analysis and filter our results. Then, we sequenced the full-length prion gene in 10 patients with sporadic CJD and characterized polymorphisms in the intronic region and the promoter of the gene. Altogether our results reveal that long-read sequencing, using MinION, allows investigating the genetic makeup of the whole PRNP in patients, including the promoter, flanking regions, intron, and both exons of the gene. We propose an accurate, quick, and user friendly method that bypasses the needs for expensive instruments and which allows for sample multiplexing. Finally, our pipeline is also able to report the results in an easily readable text format.

KEYWORDS:

Nanopore sequencing; genetic prion disease; bioinformatics; structural variants; single-nucleotide variations

References

[1] Brown K, et al. J Geriatr Psychiatry Neurol. 2010;23(4):277-298.

[2] Schmitz M, et al. Mol Neurobiol. 2017;54(6):4138-4149.

**107. Global analysis of protein degradation rates in prion infected cells by dynamic SILAC**

Charles R. Hutti, Kevin A. Welle, Jennifer R. Hryhorenko and Sina Ghaemmaghami

University of Rochester, New York

CONTACT Charles R. Hutti m.shafiq@uke.de

ABSTRACT

Prions diseases are rare neurological disorders caused by the misfolding of cellular prion protein (PrPC). The misfolded conformers aggregate into cytotoxic fibrils (PrPSc) that facilitate the conversion of additional prion proteins into the misfolded form. Intracellular PrPSc aggregates primarily
<figure><img src="image_66.png" /></figure>

accumulate within late endosomes and lysosomes, organelles that participate in the degradation and turnover of a large subset of the proteome. Thus, intracellular accumulation of PrP<sup>C</sup> aggregates may globally disrupt protein degradation kinetics within infected cells. To test this hypothesis, we analysed the effect of prion infection on protein degradation rates in N2a cells using dynamic stable isotopic labelling with amino acids in cell culture (dSILAC) and bottom-up proteomics. We were able to analyse the degradation rates of ~5000 proteins, constituting the largest proteome-wide analysis of degradation rates in prion infected cells to date. As expected, the data indicate that the degradation rate of the prion protein is significantly decreased in infected cells. Indeed, we demonstrate that cellular dilution due to cell division (rather than degradation) is the dominant factor in clearance of PrP<sup>C</sup> in infected N2a cells. Conversely, the degradation kinetics of the remainder of the N2a proteome generally increases upon infection. This effect occurs concurrently with enhancements in cellular activities of the proteasome and lysosomal hydrolases. Our data indicate that N2a cells respond to prion infection by globally upregulating the cellular protein degradation machinery. The resulting enhancement in proteome flux may play a role in the survival of N2a cells in the presence of prion infection.

**108. Crystal structural studies of Fab fragments bound to the prion molecule and perspectives on the loop connecting β2 to α2**

Pravas Kumar Baral<sup>a</sup>, Mridula Swayampakula<sup>a</sup>, Adriano Aguzzi<sup>b</sup> and Michael N. G. James<sup>a</sup>

CONTACT Michael N. G. James michael.james@ualberta.ca

ABSTRACT

Our laboratories have collaborated on determining the structures of the globular domains of prion molecules from several mammalian species by X-ray crystallography. We have used the advantage of co-crystallizing the folded domains of the cellular prion proteins (PrP<sup>C</sup>) with the Fab fragments of several immunoglobulins raised against the mouse prion. In particular, the Fab fragments of POM1 and POM6 have adjacent but slightly different binding epitopes on the mouse PrP<sup>C</sup> globular domain. Biological studies on these antibodies are quite interesting; POM1 is a highly toxic antibody whereas POM6 is an innocuous antibody. We have determined the structures

of POM1 Fab bound to the folded domains of the PrP<sup>C</sup> s of mouse, human, bovine, deer, elk, and Syrian hamster. The structure of mouse PrP<sup>C</sup> bound to the Fab fragment of the POM6 antibody has also been determined. We have compared the loop connecting the β2 strand to the α2 helix of the PrP<sup>C</sup> molecules. In all structures determined by X-ray crystallography, this loop adopts very similar structures involving a 3<sub>10</sub> helix and the α2 helix initiator residue Asn171. Another very important residue in this loop is Tyr169 and its interactions with residues on helix α2. We have compared the structures of this loop determined by X-ray crystallography to the structures of this loop as determined by NMR spectroscopy. This region differs in conformation by a substantial amount in the structures determined by the two techniques (the Ca atoms of the two Ser170 residues differ in position by 5.1Å between the X-ray structure and the NMR structure). Tighter contact between the residues of the β2-α2 loop and residues from the β1-β2 region seems to be the key in keeping the folded prion domain more stable.

**109. Analysis of N-linked glycans of ovine prion protein by liquid chromatography coupled with electrospray mass spectrometry**

Natali Nakić<sup>a</sup>, Thanh Hoa Tran<sup>a</sup>, Mislav Novokmet<sup>b</sup>, Jelena Šimunović<sup>b</sup>, Olivier Andreoletti<sup>c</sup>, Gordan Lauc<sup>b</sup> and Giuseppe Legname<sup>a</sup>

CONTACT Natali Nakić nnakic@sissa.it Laboratory of Prion Biology, Department of Neuroscience, Scuola Internazionale Superiore di Studi Avanzati (SISSA), Trieste, Italy

ABSTRACT

Prion diseases are characterized by a distinct pathology in the central nervous system, however, differing from one another in clinical symptoms, incubation time, biochemical properties, and in the deposition pattern. These differences arise from the fact that PrP<sup>C</sup> can acquire multiple conformationally different PrP<sup>Sc</sup> states, called strains. The question of how different strains display such a diversity in disease phenotypes remains to be answered. One of the ways strains are characterized is by immunoblotting, where each strain shows a specific three band pattern. The reason for this comes from the fact that the prion protein can carry up to two N-linked glycans. The knowledge about interindividual or inter prion strain differences in N-glycan structures is still not fully understood. The aim of this study is to differentiate individual N-glycan structures on different sites and to evaluate functional aspects of

<footnote><sup>a</sup>Department of Biochemistry, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Canada; <sup>b</sup>Department of Pathology, Institute of Neuropathology, University Hospital Zurich, Zurich, Switzerland</footnote>

<footnote><sup>a</sup>Laboratory of Prion Biology, Department of Neuroscience, Scuola Internazionale Superiore di Studi Avanzati (SISSA), Trieste, Italy; <sup>b</sup>Genos Glycoscience Research Laboratory, Zagreb, Croatia; <sup>c</sup>UMR INRA ENVT 1225- IHAP, École Nationale Vétérinaire de Toulouse, Toulouse, France</footnote>
<figure><img src="image_70.png" /></figure>

accumulate within late endosomes and lysosomes, organelles that participate in the degradation and turnover of a large subset of the proteome. Thus, intracellular accumulation of PrP<sup>C</sup> aggregates may globally disrupt protein degradation kinetics within infected cells. To test this hypothesis, we analysed the effect of prion infection on protein degradation rates in N2a cells using dynamic stable isotopic labelling with amino acids in cell culture (dSILAC) and bottom-up proteomics. We were able to analyse the degradation rates of ~5000 proteins, constituting the largest proteome-wide analysis of degradation rates in prion infected cells to date. As expected, the data indicate that the degradation rate of the prion protein is significantly decreased in infected cells. Indeed, we demonstrate that cellular dilution due to cell division (rather than degradation) is the dominant factor in clearance of PrP<sup>C</sup> in infected N2a cells. Conversely, the degradation kinetics of the remainder of the N2a proteome generally increases upon infection. This effect occurs concurrently with enhancements in cellular activities of the proteasome and lysosomal hydrolases. Our data indicate that N2a cells respond to prion infection by globally upregulating the cellular protein degradation machinery. The resulting enhancement in proteome flux may play a role in the survival of N2a cells in the presence of prion infection.

**108. Crystal structural studies of Fab fragments bound to the prion molecule and perspectives on the loop connecting β2 to α2**

Pravas Kumar Baral<sup>a</sup>, Mridula Swayampakula<sup>a</sup>, Adriano Aguzzi<sup>b</sup> and Michael N. G. James<sup>a</sup>

CONTACT Michael N. G. James michael.james@ualberta.ca

**ABSTRACT**

Our laboratories have collaborated on determining the structures of the globular domains of prion molecules from several mammalian species by X-ray crystallography. We have used the advantage of co-crystallizing the folded domains of the cellular prion proteins (PrP<sup>C</sup>) with the Fab fragments of several immunoglobulins raised against the mouse prion. In particular, the Fab fragments of POM1 and POM6 have adjacent but slightly different binding epitopes on the mouse PrP<sup>C</sup> globular domain. Biological studies on these antibodies are quite interesting; POM1 is a highly toxic antibody whereas POM6 is an innocuous antibody. We have determined the structures

of POM1 Fab bound to the folded domains of the PrP<sup>C</sup> s of mouse, human, bovine, deer, elk, and Syrian hamster. The structure of mouse PrP<sup>C</sup> bound to the Fab fragment of the POM6 antibody has also been determined. We have compared the loop connecting the β2 strand to the α2 helix of the PrP<sup>C</sup> molecules. In all structures determined by X-ray crystallography, this loop adopts very similar structures involving a 3<sub>10</sub> helix and the α2 helix initiator residue Asn171. Another very important residue in this loop is Tyr169 and its interactions with residues on helix α2. We have compared the structures of this loop determined by X-ray crystallography to the structures of this loop as determined by NMR spectroscopy. This region differs in conformation by a substantial amount in the structures determined by the two techniques (the Ca atoms of the two Ser170 residues differ in position by 5.1Å between the X-ray structure and the NMR structure). Tighter contact between the residues of the β2-α2 loop and residues from the β1-β2 region seems to be the key in keeping the folded prion domain more stable.

**109. Analysis of N-linked glycans of ovine prion protein by liquid chromatography coupled with electrospray mass spectrometry**

Natali Nakić<sup>a</sup>, Thanh Hoa Tran<sup>a</sup>, Mislav Novokmet<sup>b</sup>, Jelena Šimunović<sup>b</sup>, Olivier Andreoletti<sup>c</sup>, Gordan Lauc<sup>b</sup> and Giuseppe Legname<sup>a</sup>

CONTACT Natali Nakić nnakic@sissa.it Laboratory of Prion Biology, Department of Neuroscience, Scuola Internazionale Superiore di Studi Avanzati (SISSA), Trieste, Italy

**ABSTRACT**

Prion diseases are characterized by a distinct pathology in the central nervous system, however, differing from one another in clinical symptoms, incubation time, biochemical properties, and in the deposition pattern. These differences arise from the fact that PrP<sup>C</sup> can acquire multiple conformationally different PrP<sup>C</sup> states, called strains. The question of how different strains display such a diversity in disease phenotypes remains to be answered. One of the ways strains are characterized is by immunoblotting, where each strain shows a specific three band pattern. The reason for this comes from the fact that the prion protein can carry up to two N-linked glycans. The knowledge about interindividual or inter prion strain differences in N-glycan structures is still not fully understood. The aim of this study is to differentiate individual N-glycan structures on different sites and to evaluate functional aspects of

<footnote><sup>a</sup>Department of Biochemistry, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Canada; <sup>b</sup>Department of Pathology, Institute of Neuropathology, University Hospital Zurich, Zurich, Switzerland</footnote>

<footnote><sup>a</sup>Laboratory of Prion Biology, Department of Neuroscience, Scuola Internazionale Superiore di Studi Avanzati (SISSA), Trieste, Italy; <sup>b</sup>Genos Glycoscience Research Laboratory, Zagreb, Croatia; <sup>c</sup>UMR INRA ENVT 1225- IHAP, École Nationale Vétérinaire de Toulouse, Toulouse, France</footnote>