<figure><img src="image_111.png" /></figure>

(PrPCWD) shed by infected animals. Prion fate in environment is determined by structure of ecosystems including soil and vegetation types. In boreal ecosystems, lichen and shrubs cover sandy textured, quartz-illite soils with surface plant litter horizon. In prairie ecosystems, grassy meadows grow on humus-enriched, loamy or clay montmorillonite soils. Once shed onto the soil surface, prion persistence in the environment primarily depends on the binding and transportation capacity of soils. Factors responsible for prion migration are soil properties such as mineralogical composition, texture, soil organic matter content and pH. We investigated PrPCWD migration in soil profiles by comparing their passage through pure soil minerals and diverse soils using lab-scale soil columns. PrP-res was detected by western blot or protein misfolding cyclic amplification in leachates of columns containing quartz, surface plant litter - soil organic horizon (LFH) of boreal Luvisolic and Brunisolic soils, illite, or the mineral horizons of Luvisol and Brunisol. Infectivity in leachates from quartz, illite and Luvisolic columns was confirmed by bioassay. Analysis of the solid phase of the columns confirmed the migration of PrPCWD to lower layers in the illite, while the PrPCWD in the montmorillonite column remained close to the column surface. While mineralogical composition of soils determines movement of prions, other constituents, such as soil organic matter, microbial population, composition of soil solutions, might affect prion infectivity. To investigate the impact of humic acids (a major component of soil organic material), we incubated CWD agent with humic acids for various lengths of time. Analysis by western blot and bioassay demonstrated that humic acids can reduce PrPres and infectivity over time. Thus, in soils of temperate regions, including the Chernozems of Northern America and Cambisols of Europe, prions would remain at the soil surface due to strong binding to montmorillonite, while in boreal and tundra regions soils (Luvisols, Podzols, and Brunisols), most of the prions would be transported through the plant litter LFH, and partially through the upper soil mineral horizon into lower horizons. Although these soils also have lower levels of humic acids, a soil organic matter compound that has the capacity to reduce CWD infectivity levels, soils of the boreal and tundra regions are less favourable to the horizontal transmission of CWD as these soils make PrPCWD less bioavailable.

**176. Ultrasensitive detection and seeding selectivity of tau aggregates of Alzheimer disease and chronic traumatic encephalopathy**

Allison Kraus<sup>a</sup>, Eri Saijo<sup>a</sup>, Michael M. Metrick<sup>a</sup>, Kathy Newell<sup>b</sup>, Christina J. Sigurdson<sup>c</sup>, Gianluigi Zanusso<sup>d</sup>, Bernardino Ghetti<sup>e</sup> and Byron Caughey<sup>a</sup>

<sup>a</sup>LPVD, Rocky Mountain Laboratories, NIAID, NIH, Hamilton, MT, USA;

<sup>b</sup>University of Kansas School of Medicine, Kansas City, KS, USA;

<sup>c</sup>Department of Pathology, UC San Diego, La Jolla, CA, USA;

<sup>d</sup>University of Verona, Verona, Italy; <sup>e</sup>Indiana University School of Medicine, Indianapolis, IN, USA

CONTACT Allison Kraus allison.kraus@nih.gov

**ABSTRACT**

In Alzheimer disease (AD) and chronic traumatic encephalopathy (CTE) approximately equivalent amounts of 3-repeat (3R) and 4-repeat (4R) tau isoforms accumulate in tau filaments in the brain. To investigate the basis for AD 3R/4R tau filament propagation and to improve detection of 3R/4R tau aggregates as potential biomarkers, we have exploited the growth mechanism of tau filaments to develop a highly selective and ultrasensitive cell-free tau seed amplification assay optimized for AD (AD real-time quaking induced conversion or AD RT-QuIC). This reaction is based on the capacity of AD tau aggregates to seed formation of amyloids made of certain tau fragments. Seeding activity was detected in AD brains (n = 16) at dilutions as extreme as 10⁷–10¹⁰- fold but was 10²–10⁶-fold less responsive when seeded with brain from most cases of other types of tauopathy with roughly comparable loads of tau aggregates composed predominantly of either 3R or 4R tau isoforms. AD brains had average seeding activities that were orders of magnitude higher than Pick disease brains with predominant 3R tau deposits; however, we previously observed the opposite using our prototypic Pick- optimized tau RT-QuIC assay. CTE brains (n = 2) had seed concentrations comparable to some of the AD specimens, and higher than three of four specimens with 3R/4R primary age-related tauopathy (i.e., PART). AD seeds shared properties with the neurofibrillary tangles found in AD brains in terms of being sarkosyl-insoluble, protease-resistant, and reactive with tau antibodies.

Moreover, the AD RT-QuIC assay detected as little as 16 fg of pure synthetic tau fibrils. The distinctive seeding activity shown by AD and CTE tau filaments in comparison to those of other types of tauopathies suggests a templating specificity that may underpin their consistent strain- like propagation as conformers in patients with 3R/4R tau diseases. Importantly, AD RT-QuIC also provides a means of rapid and ultrasensitive quantitation of 3R/4R tau seeding activity as a biomarker. We are currently adapting the assay to the detection of AD and CTE tau seeds in
<figure><img src="image_111.png" /></figure>

(PrPCWD) shed by infected animals. Prion fate in environment is determined by structure of ecosystems including soil and vegetation types. In boreal ecosystems, lichen and shrubs cover sandy textured, quartz-illite soils with surface plant litter horizon. In prairie ecosystems, grassy meadows grow on humus-enriched, loamy or clay montmorillonite soils. Once shed onto the soil surface, prion persistence in the environment primarily depends on the binding and transportation capacity of soils. Factors responsible for prion migration are soil properties such as mineralogical composition, texture, soil organic matter content and pH. We investigated PrPCWD migration in soil profiles by comparing their passage through pure soil minerals and diverse soils using lab-scale soil columns. PrP-res was detected by western blot or protein misfolding cyclic amplification in leachates of columns containing quartz, surface plant litter - soil organic horizon (LFH) of boreal Luvisolic and Brunisolic soils, illite, or the mineral horizons of Luvisol and Brunisol. Infectivity in leachates from quartz, illite and Luvisolic columns was confirmed by bioassay. Analysis of the solid phase of the columns confirmed the migration of PrPCWD to lower layers in the illite, while the PrPCWD in the montmorillonite column remained close to the column surface. While mineralogical composition of soils determines movement of prions, other constituents, such as soil organic matter, microbial population, composition of soil solutions, might affect prion infectivity. To investigate the impact of humic acids (a major component of soil organic material), we incubated CWD agent with humic acids for various lengths of time. Analysis by western blot and bioassay demonstrated that humic acids can reduce PrPres and infectivity over time. Thus, in soils of temperate regions, including the Chernozems of Northern America and Cambisols of Europe, prions would remain at the soil surface due to strong binding to montmorillonite, while in boreal and tundra regions soils (Luvisols, Podzols, and Brunisols), most of the prions would be transported through the plant litter LFH, and partially through the upper soil mineral horizon into lower horizons. Although these soils also have lower levels of humic acids, a soil organic matter compound that has the capacity to reduce CWD infectivity levels, soils of the boreal and tundra regions are less favourable to the horizontal transmission of CWD as these soils make PrPCWD less bioavailable.

## 176. Ultrasensitive detection and seeding selectivity of tau aggregates of Alzheimer disease and chronic traumatic encephalopathy

Allison Krausª, Eri Saijoª, Michael M. Metrickª, Kathy Newellᵇ, Christina J. Sigurdsonᶜ, Gianluigi Zanussoᵈ, Bernardino Ghettiᵉ and Byron Caugheyª

ªLPVD, Rocky Mountain Laboratories, NIAID, NIH, Hamilton, MT, USA;

ᵇUniversity of Kansas School of Medicine, Kansas City, KS, USA;

ᶜDepartment of Pathology, UC San Diego, La Jolla, CA, USA;

ᵈUniversity of Verona, Verona, Italy; eIndiana University School of Medicine, Indianapolis, IN, USA

CONTACT Allison Kraus allison.kraus@nih.gov

## ABSTRACT

In Alzheimer disease (AD) and chronic traumatic encephalopathy (CTE) approximately equivalent amounts of 3-repeat (3R) and 4-repeat (4R) tau isoforms accumulate in tau filaments in the brain. To investigate the basis for AD 3R/4R tau filament propagation and to improve detection of 3R/4R tau aggregates as potential biomarkers, we have exploited the growth mechanism of tau filaments to develop a highly selective and ultrasensitive cell-free tau seed amplification assay optimized for AD (AD real-time quaking induced conversion or AD RT-QuIC). This reaction is based on the capacity of AD tau aggregates to seed formation of amyloids made of certain tau fragments. Seeding activity was detected in AD brains (n = 16) at dilutions as extreme as 10⁷–10¹⁰-fold but was 10²–10⁶-fold less responsive when seeded with brain from most cases of other types of tauopathy with roughly comparable loads of tau aggregates composed predominantly of either 3R or 4R tau isoforms. AD brains had average seeding activities that were orders of magnitude higher than Pick disease brains with predominant 3R tau deposits; however, we previously observed the opposite using our prototypic Pick- optimized tau RT-QuIC assay. CTE brains (n = 2) had seed concentrations comparable to some of the AD specimens, and higher than three of four specimens with 3R/4R primary age-related tauopathy (i.e., PART). AD seeds shared properties with the neurofibrillary tangles found in AD brains in terms of being sarkosyl-insoluble, protease-resistant, and reactive with tau antibodies.

Moreover, the AD RT-QuIC assay detected as little as 16 fg of pure synthetic tau fibrils. The distinctive seeding activity shown by AD and CTE tau filaments in comparison to those of other types of tauopathies suggests a templating specificity that may underpin their consistent strain- like propagation as conformers in patients with 3R/4R tau diseases. Importantly, AD RT-QuIC also provides a means of rapid and ultrasensitive quantitation of 3R/4R tau seeding activity as a biomarker. We are currently adapting the assay to the detection of AD and CTE tau seeds in
<figure><img src="image_112.png" /></figure>

biologically accessible specimens to aid in diagnostics and therapeutics development.

**KEYWORDS:** Alzheimer disease; chronic traumatic encephalopathy; tau; seed; diagnostics; biomarkers; RT-QuIC

## Reference

[1] Kraus A et al., Acta Neuropath. 2018:[Epub ahead of print].

# 177. Assessing the importance of Tau mislocalization to the dendrites in Alzheimer's disease

Emily A. McNamara, Sue-Ann Mok

Department of Biochemistry, University of Alberta, Edmonton, Canada

CONTACT Emily A. McNamara emcnamar@ualberta.ca

## ABSTRACT

Alzheimer's disease is a fatal neurodegenerative condition primarily characterized by a decline in cognitive function and a loss of motor skills¹. This disease affects cells of the central nervous system (neurons) and is the leading cause of all dementias¹. While the exact cause of Alzheimer's disease is not yet fully understood, one of the main components thought to be associated with disease pathology is the abnormal accumulation of the protein, Tau². Tau is a microtubule-associated protein found primarily in the neuronal axon, where it plays a role in assembling microtubules that are critical for axonal transport³. Studies have shown that Tau mislocalization to the dendritic spines can occur during or preceding a disease state, causing synaptic disruption and overall brain dysfunction⁴. Specific signals in the 3' Untranslated Region (UTR) of Tau mRNA transcripts have been shown to play a key role in subcellular mRNA localization and therefore local protein translation⁵.

Our work aims to modify the human Tau 3' UTR in order to localize it to the neuronal axon or dendritic spines. Following this, we will be able to explore Tau's interactions with various cellular components at either subcellular location and potentially uncover key components involved in disease pathology.

We have designed constructs of the Tau 0N4R isoform with a modified 3' UTR. The 3' UTR's utilized are derived from human Tau or mouse Tau, which localize to the axon, or another microtubule associated protein, MAP2, which localizes to the dendrites. While it has been shown that mouse Tau 3' UTR is capable of directing Tau to the

axons⁵, this has not yet been replicated with human Tau. The constructs will be transfected into human neuronal cells using lentiviral vectors. Fluorescent antibodies and mRNA probes recognizing specific regions of exogenously-expressed Tau will be used to detect the correct localization of the protein and transcript. While the analysis of Tau interactions at both the axon and dendrites are part of my long-term project, HEK cell lines that express Tau 0N4R have been transfected with DNAJ2 and Hsc70; two chaperones known to be upregulated during Alzheimer's Disease. HEK cells will be induced to express Tau, and potential aggregation will be monitored using confocal microscopy. Results from this assay may pinpoint DNAJ2 and Hsc70 as key players in Tau aggregation, and results will be replicated in a human neuronal model.

Understanding the importance of Tau localization in neurodegenerative disease pathology may uncover potential key therapeutic targets for the treatment of Alzheimer's disease.

**KEYWORDS:** Alzheimer's disease; Tau; neurodegeneration; 3' UTR

## References

[1] Alzheimer's Association. 2015 Alzheimer's Disease facts and figures. Alzheimer's & Dementia. 2015;11 (2015):332-384.

[2] Musi N., Valentine JM. et al., Tau protein aggregation is associated with cellular senescence in the brain. Wiley Aging Cell. 2018:1-13. DOI:10.1111/accel.12,840

[3] Buée L. et al., Tau protein isofroms, phosphorylation and role in neurodegenerative disorders. Brain Research Reviews. 2000;33:95-130.

[4] Hoover BR. et al., Tau mislocalization to dendritic spines mediates synaptic dysfunction independently of neurodegeneration. Cell Press Neuron. 2010;68:1067-1081.

[5] Aronov S. et al., Axonal Tau mRNA localization coincides with tau protein in living neuronal cells and depends on axonal targeting signal. The Journal of Neuroscience. 2001;21(17):6577-6587.

# 178. Distinct strain of Amyloid beta and pathogenic tau protein in iatrogenic Creutzfeldt-Jakob disease

Jiri G. Safarª, Chae Kimª, Tracy Haldimanª, Anushruti Ashokaª, Ignazio Caliª, Mark Cohenª, Stéphane Haïkb, Piero Parchic, Giorgio Giacconeq, Steven Collinsr, Nicolas Privatb, Véronique Sazdovitchb, Charles Duyckaertsb, Diane Kofskeyª, Han Wangª, Catriona McLeane, Jean-Philippe Brandelb, Tetsuyuki Kitamotof, Ermias Belayg, Ryan Maddoxh, Fabrizio Tagliavini d, Maurizio Pocchiar i, Brian Applebya,
<figure><img src="image_112.png" /></figure>

biologically accessible specimens to aid in diagnostics and therapeutics development.

**KEYWORDS:** Alzheimer disease; chronic traumatic encephalopathy; tau; seed; diagnostics; biomarkers; RT-QuIC

## Reference

[1] Kraus A et al., Acta Neuropath. 2018:[Epub ahead of print].

# 177. Assessing the importance of Tau mislocalization to the dendrites in Alzheimer's disease

Emily A. McNamara, Sue-Ann Mok

Department of Biochemistry, University of Alberta, Edmonton, Canada

CONTACT Emily A. McNamara emcnamar@ualberta.ca

## ABSTRACT

Alzheimer's disease is a fatal neurodegenerative condition primarily characterized by a decline in cognitive function and a loss of motor skills¹. This disease affects cells of the central nervous system (neurons) and is the leading cause of all dementias¹. While the exact cause of Alzheimer's disease is not yet fully understood, one of the main components thought to be associated with disease pathology is the abnormal accumulation of the protein, Tau². Tau is a microtubule-associated protein found primarily in the neuronal axon, where it plays a role in assembling microtubules that are critical for axonal transport³. Studies have shown that Tau mislocalization to the dendritic spines can occur during or preceding a disease state, causing synaptic disruption and overall brain dysfunction⁴. Specific signals in the 3' Untranslated Region (UTR) of Tau mRNA transcripts have been shown to play a key role in subcellular mRNA localization and therefore local protein translation⁵.

Our work aims to modify the human Tau 3' UTR in order to localize it to the neuronal axon or dendritic spines. Following this, we will be able to explore Tau's interactions with various cellular components at either subcellular location and potentially uncover key components involved in disease pathology.

We have designed constructs of the Tau 0N4R isoform with a modified 3' UTR. The 3' UTR's utilized are derived from human Tau or mouse Tau, which localize to the axon, or another microtubule associated protein, MAP2, which localizes to the dendrites. While it has been shown that mouse Tau 3' UTR is capable of directing Tau to the

axons⁵, this has not yet been replicated with human Tau. The constructs will be transfected into human neuronal cells using lentiviral vectors. Fluorescent antibodies and mRNA probes recognizing specific regions of exogenously-expressed Tau will be used to detect the correct localization of the protein and transcript. While the analysis of Tau interactions at both the axon and dendrites are part of my long-term project, HEK cell lines that express Tau 0N4R have been transfected with DNAJ2 and Hsc70; two chaperones known to be upregulated during Alzheimer's Disease. HEK cells will be induced to express Tau, and potential aggregation will be monitored using confocal microscopy. Results from this assay may pinpoint DNAJ2 and Hsc70 as key players in Tau aggregation, and results will be replicated in a human neuronal model.

Understanding the importance of Tau localization in neurodegenerative disease pathology may uncover potential key therapeutic targets for the treatment of Alzheimer's disease.

**KEYWORDS:** Alzheimer's disease; Tau; neurodegeneration; 3' UTR

## References

[1] Alzheimer's Association. 2015 Alzheimer's Disease facts and figures. Alzheimer's & Dementia. 2015;11 (2015):332-384.

[2] Musi N., Valentine JM. et al., Tau protein aggregation is associated with cellular senescence in the brain. Wiley Aging Cell. 2018:1-13. DOI:10.1111/ace.12,840

[3] Buée L. et al., Tau protein isofroms, phosphorylation and role in neurodegenerative disorders. Brain Research Reviews. 2000;33:95-130.

[4] Hoover BR. et al., Tau mislocalization to dendritic spines mediates synaptic dysfunction independently of neurodegeneration. Cell Press Neuron. 2010;68:1067-1081.

[5] Aronov S. et al., Axonal Tau mRNA localization coincides with tau protein in living neuronal cells and depends on axonal targeting signal. The Journal of Neuroscience. 2001;21(17):6577-6587.

# 178. Distinct strain of Amyloid beta and pathogenic tau protein in iatrogenic Creutzfeldt-Jakob disease

Jiri G. Safarª, Chae Kimª, Tracy Haldimanª, Anushruti Ashokaª, Ignazio Caliª, Mark Cohenª, Stéphane Haïkb, Piero Parchic, Giorgio Giacconeq, Steven Collinsr, Nicolas Privatb, Véronique Sazdovitchb, Charles Duyckaertsb, Diane Kofskeyª, Han Wangª, Catriona McLeane, Jean-Philippe Brandelb, Tetsuyuki Kitamotof, Ermias Belayg, Ryan Maddoxh, Fabrizio Tagliavini d, Maurizio Pocchiar i, Brian Applebya,
<figure><img src="image_113.png" /></figure>

Pierluigi Gambetti<sup>j</sup>, Ele Gelpi<sup>k</sup>, Ann McKee<sup>l</sup>, James Ironside<sup>m</sup> and Lawrence Schonberger<sup>n</sup>

<sup>a</sup>Case Western Reserve University; <sup>b</sup>Sorbonne Universités; <sup>c</sup>University of Bologna; <sup>d</sup>Istituto Neurologico Carlo Besta; <sup>e</sup>University of Melbourne; <sup>f</sup>Tohoku University; <sup>g</sup>Center for Disease Control and Prevention; <sup>h</sup>CDC; <sup>i</sup>Istituto Superiore di Sanità; <sup>j</sup>Department of Pathology, Case Western Reserve University, School of Medicine, Cleveland, OH, USA; <sup>k</sup>Hospital Clínic Institut d'Investigacions Biomèdiques August Pi i Sunyer; <sup>l</sup>Boston University; <sup>m</sup>University of Edinburgh; <sup>n</sup>US Centre for Disease Control and Prevention

ABSTRACT

**Objectives:** Whether some strains of prion-like protein aggregates (prionoids) such as amyloid beta (Aβ) and tau propagating in cell culture and transgenic experiments can be infectious and transmit the neurodegenerative diseases from man-to-man is unknown. We reported recently an increased frequency of amyloid β (Aβ) plaques and angiopathy in a cohort of 27 iatrogenic cases of Creutzfeldt-Jakob disease (iCJD), acquired from the prion-contaminated growth hormone (GH) extracted from human cadaveric pituitary glands, or from cadaveric dura mater grafts (DM) during neurosurgery. As in human prion diseases, critical questions raised by these findings are (a) whether the pathology was transmitted by a unique strain of Aβ or tau with a high potential to initiate or accelerate Alzheimer's disease (AD), and (b) if such Aβ or tau strains can be identified within the spectrum of AD.

**Methods:** With new biophysical and structural tools, we analysed conformational strain characteristics of Aβ and pathogenic tau aggregates in 14 cases of iCJD, and compared the data with 21 cases of age-matched sporadic CJD (sCJD), 30 cases of Alzheimer's diseases with rapid progression (rpAD), 18 cases with slow AD progression (spAD), 7 cases of chronic traumatic encephalopathy (CTE), and 8 cases of frontotemporal lobar degeneration (FTLD).

**Results:** The levels of oligomeric and aggregated Aβ42 as well as the Aβ42/Aβ40 ratios were in iCJD cases elevated significantly above the levels found in sCJD controls. The conformational characteristics of Aβ42 accumulating in iCJD were distinctly different from sCJD and reminiscent of small oligomers of Aβ42 found most frequently in AD with rapid progression. The levels of detergent-insoluble pathogenic tau aggregates were increased in ~14% of iCJD above the levels found in sCJD but were generally lower than in AD and other tauopathies. Notably, the conformations of detergent-insoluble tau found in hippocampus of iCJD cases were significantly different from those found in sCJD.

**Conclusions:** The levels of oligomeric and aggregated forms of Ab42 and pathogenic aggregates of tau protein are in iCJD cases significantly higher than in sCJD

controls and their conformational characteristics are in the range corresponding to the strains found most frequently in rapidly progressive AD. Although the contributions of other factors, including GH deficiency cannot be discounted, our findings argue that this AD pathology was acquired by a distinct strain of amyloid beta conformers through a mechanism resembling that of prion diseases.

179. PrP<sup>CWD</sup> detection in CWD-infected TgElk mice model using RT-QUIC

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

Foreign animal disease division, Animal and Plant Quarantine Agency, Gimcheon, Gyeongsangbuk-do, Korea

ABSTRACT

**Introduction:** Chronic wasting disease (CWD) is the only prion disease affecting free-ranging animals, reported in North America, South Korea, and Norway. Unlike in most other prion disease CWD agents are shed in blood, urine, and faeces which most likely contribute to the horizontal transmission between cervid species. The developments of amplification-based seeding assays have been instrumental in the detection of low levels of prions in clinical samples. Using real-time quaking-induced conversion (RT-QUIC), we established an ultrasensitive detection method for PrP<sup>CWD</sup> in the urine from CWD-infected sequentially sampled transgenic mice overexpressing elk prion protein (TgElk mice). In addtion, RT-QUIC was performed in the kidney and brain of theses mice model to trace abnormal prion.

**Materials and Methods:** 44 brain and kidney, urine samples from sequentially collected from CWD-infected TgElk mice (TgElk CWD) were stored at -80°C. In brain and kidney, 10% (w/v) homogenate was prepared in 0.9% sterilized saline. In urine 100 µL of each sample was mixed with 10 µL 2.8% sodium phosphotungustic acid (NaPTA) and incubated for 1hr at 37°C with shaking at 1,350 rpm. Samples were centrifuged for 30 min at 16,100 g. The pellet was resuspended in 10 µL of 0.1% SDS/PBS for 30 min at 55°C. RT-QUIC reactions were set up in 96-well clear bottom optic plates and consisted of 98 µL RT-QUIC buffer [final concentrations of 1XPBS, 1 mM EDTA, 10 µM Thioflavin, 300 mM NaCl buffer and 0.1 mg/ml recombinant Syrian hamster recombinant protein (23-231), and 2 µL of sample. The RT-QUIC assay was performed on a FLUOstar Omega fluorescence plate reader that was preheated to 42°C for 60 h with 90 s shaking at 700 rpm followed by 1 min incubation.
<figure><img src="image_113.png" /></figure>

Pierluigi Gambetti<sup>j</sup>, Ele Gelpi<sup>k</sup>, Ann McKee<sup>l</sup>, James Ironside<sup>m</sup> and Lawrence Schonberger<sup>n</sup>

<sup>a</sup>Case Western Reserve University; <sup>b</sup>Sorbonne Universités; <sup>c</sup>University of Bologna; <sup>d</sup>Istituto Neurologico Carlo Besta; <sup>e</sup>University of Melbourne; <sup>f</sup>Tohoku University; <sup>g</sup>Center for Disease Control and Prevention; <sup>h</sup>CDC; <sup>i</sup>Istituto Superiore di Sanità; <sup>j</sup>Department of Pathology, Case Western Reserve University, School of Medicine, Cleveland, OH, USA; <sup>k</sup>Hospital Clínico Institut d'Investigacions Biomèdiques August Pi i Sunyer; <sup>l</sup>Boston University; <sup>m</sup>University of Edinburgh; <sup>n</sup>US Centre for Disease Control and Prevention

ABSTRACT

**Objectives:** Whether some strains of prion-like protein aggregates (prionoids) such as amyloid beta (Aβ) and tau propagating in cell culture and transgenic experiments can be infectious and transmit the neurodegenerative diseases from man-to-man is unknown. We reported recently an increased frequency of amyloid β (Aβ) plaques and angiopathy in a cohort of 27 iatrogenic cases of Creutzfeldt-Jakob disease (iCJD), acquired from the prion-contaminated growth hormone (GH) extracted from human cadaveric pituitary glands, or from cadaveric dura mater grafts (DM) during neurosurgery. As in human prion diseases, critical questions raised by these findings are (a) whether the pathology was transmitted by a unique strain of Aβ or tau with a high potential to initiate or accelerate Alzheimer's disease (AD), and (b) if such Aβ or tau strains can be identified within the spectrum of AD.

**Methods:** With new biophysical and structural tools, we analysed conformational strain characteristics of Aβ and pathogenic tau aggregates in 14 cases of iCJD, and compared the data with 21 cases of age-matched sporadic CJD (sCJD), 30 cases of Alzheimer's diseases with rapid progression (rpAD), 18 cases with slow AD progression (spAD), 7 cases of chronic traumatic encephalopathy (CTE), and 8 cases of frontotemporal lobar degeneration (FTLD).

**Results:** The levels of oligomeric and aggregated Aβ42 as well as the Aβ42/Aβ40 ratios were in iCJD cases elevated significantly above the levels found in sCJD controls. The conformational characteristics of Aβ42 accumulating in iCJD were distinctly different from sCJD and reminiscent of small oligomers of Aβ42 found most frequently in AD with rapid progression. The levels of detergent-insoluble pathogenic tau aggregates were increased in ~14% of iCJD above the levels found in sCJD but were generally lower than in AD and other tauopathies. Notably, the conformations of detergent-insoluble tau found in hippocampus of iCJD cases were significantly different from those found in sCJD.

**Conclusions:** The levels of oligomeric and aggregated forms of Ab42 and pathogenic aggregates of tau protein are in iCJD cases significantly higher than in sCJD

controls and their conformational characteristics are in the range corresponding to the strains found most frequently in rapidly progressive AD. Although the contributions of other factors, including GH deficiency cannot be discounted, our findings argue that this AD pathology was acquired by a distinct strain of amyloid beta conformers through a mechanism resembling that of prion diseases.

179. PrP<sup>CWD</sup> detection in CWD-infected TgElk mice model using RT-QUIC

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

Foreign animal disease division, Animal and Plant Quarantine Agency, Gimcheon, Gyeongsangbuk-do, Korea

ABSTRACT

**Introduction:** Chronic wasting disease (CWD) is the only prion disease affecting free-ranging animals, reported in North America, South Korea, and Norway. Unlike in most other prion disease CWD agents are shed in blood, urine, and faeces which most likely contribute to the horizontal transmission between cervid species. The developments of amplification-based seeding assays have been instrumental in the detection of low levels of prions in clinical samples. Using real-time quaking-induced conversion (RT-QUIC), we established an ultrasensitive detection method for PrP<sup>CWD</sup> in the urine from CWD-infected sequentially sampled transgenic mice overexpressing elk prion protein (TgElk mice). In addtion, RT-QUIC was performed in the kidney and brain of theses mice model to trace abnormal prion.

**Materials and Methods:** 44 brain and kidney, urine samples from sequentially collected from CWD-infected TgElk mice (TgElk CWD) were stored at -80°C. In brain and kidney, 10% (w/v) homogenate was prepared in 0.9% sterilized saline. In urine 100 µL of each sample was mixed with 10 µL 2.8% sodium phosphotungustic acid (NaPTA) and incubated for 1hr at 37°C with shaking at 1,350 rpm. Samples were centrifuged for 30 min at 16,100 g. The pellet was resuspended in 10 µL of 0.1% SDS/PBS for 30 min at 55°C. RT-QUIC reactions were set up in 96-well clear bottom optic plates and consisted of 98 µL RT-QUIC buffer [final concentrations of 1XPBS, 1 mM EDTA, 10 µM Thioflavin, 300 mM NaCl buffer and 0.1 mg/ml recombinant Syrian hamster recombinant protein (23-231), and 2 µL of sample. The RT-QUIC assay was performed on a FLUOstar Omega fluorescence plate reader that was preheated to 42°C for 60 h with 90 s shaking at 700 rpm followed by 1 min incubation.
<figure><img src="image_114.png" /></figure>

**Results:** Five randomly selected mice were sequentially culled on every 15 days from 30dpi to 120dpi during CWD infected TgElk mice reached terminal stage. Rough hair coats among clinical signs were showed from 90 dpi. $PrP^{CWD}$ in the brain in TgElk CWD was detectable persistently from early stages (30dpi), and in the kidney $PrP^{CWD}$ was also detectable in clinical and terminal stages (90 dpi and 120dpi). $PrP^{CWD}$ in the urine in TgElk CWD reached the highest levels at 120dpi. NaPTA/RT-QUIC was applied to measure $PrP^{CWD}$ in urine samples collected on every 15 days from 30dpi to 120dpi when CWD infected TgElk mice reached terminal stage. $PrP^{CWD}$ in the urine in TgElk CWD reached the highest levels at 90dpi. $PrP^{CWD}$ was also detectable in late and terminal stages (120dpi).

**Conclusions:** We demonstrate that CWD prions can be detected by RT-QUIC or NaPTA/RT-QUIC in the brain, kidney and urine of TgElk mice at the early and terminal stages of disease. Based on these data, we suggest that $PrP^{CWD}$ is excreted into only urine until 90 dpi and then slowly accumulated in kidney. Our results can be used in designing future study of CWD pathogenesis in TgElk mice.

## References

[1] Henderson DM et al., Rapid antemortem detection of CWD prions in deer saliva. PLOS one. 2013:e74377 1-12

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

## 180. Cell assay detection of chronic wasting disease (CWD) prion infectivity

Su Bi Ahnª, Hyo-Jin Kimª, Jifeng Bianᵇ, In-Soon Rohª, Hae-Eun Kangª, Hyun-Joo Sohnª

**CONTACT** Hyun-Joo Sohn

### ABSTRACT

Chronic wasting disease (CWD) is a prion disease affecting the animals in family Cervidae. Prions are the infectious agents that cause prion disease. Mouse prion bioassay is the most commonly used method for determine prion infectivity. Application of more efficient measurement method was required as several cases of CWD have been reported in South Korea. In this study, we adapted the modified cervid prion cell assay (CPCA) using $Elk21^-$ cells. The susceptible

$Elk21^-$ cells were exposed to various CWD brain homogenates, ranging from $10^{-2}$ to $10^{-5}$. The CPCA titers of elk CWD brain homogenates were $10^{5.8}$ [2001 (E190Y+229Y)] and $10^{6.5}$ [16FC050] units/g of brain, respectively. We also found the CWD prion titer of red and sika deer brain inocula in 2016 CWD cases, producing CPCA titers of $10^{5.8}$ and $10^{5.4}$ units/g of brain. These results were confirmed by western blotting. Comparing mouse titers of mouse ic LD50/g of cervid brain with CPCA titers, sensitivity of the cell-based measurement was similar to that of the mouse bioassay. Also, CPCA titers does not depend on CWD species, relate with mouse bioassay survival time. Consequently, our results demonstrate that CWD prion infectivity can be efficiently and quantitatively detected by cell based titration of CWD.

## 181. Application of the TSE-infected cell and RT-QUIC assay to discover novel natural products

Hyo-Jin Kim, Su Bi Ahn, In-Soon Roh, Kyung-Je Park, Hae-Eun Kang, Hyun-Joo Sohn

Foreign Animal Disease Division, Animal and Plant Quarantine Agency, Gimcheon, Gyeongsangbukdo, Korea

**CONTACT** Hyun-Joo Sohn

### ABSTRACT

Conformational conversion of the normal prion protein ($PrP^C$) into an infectious $PrP^{Sc}$ causes pathogenesis in prion diseases or transmissible spongiform encephalopathies (TSEs). TSE-infected cell lines have been efficiently used to study this conversion process, as well as to screen for potentially effective anti-prion compounds. We employed fragment molecular orbital (FMO)-based virtual screening and BSE-infected cell-based assay system to discover two natural products with antiprion activity exhibited good binding interactions, with hotspot residues within the $PrP^C$ binding site, and effectively reduced $PrP^{Sc}$ levels in a standard scrapie cell assay (SSCA). We also investigated the prion inhibition ability of two novel natural products in Elk $21^+$ (CWD) or SMB (Scrapie) cells. Cytotoxicity tests showed no significant cell death in either BNP-03 or BNP-08 treatment. Elk $21^+$ and SMB cells were subjected to two effective natural products for reducing $PrP^{Sc}$, BNP-03, and BNP-08. Efficiency of $PrP^{Sc}$ clearance for two natural products was estimated by evaluating the signal sensitivity for $PrP^{Sc}$ on SSCA, WB, and EIA. Antiprion activity of the compounds was detected by RT-QUIC assay seeded with TgElk CWD (E190Y +229Y). These natural products showed the inhibitory effects in the TSE-infected cells. Further experiments in

<footnote>ªForeign Animal Disease Division, Animal and Plant Quarantine Agency, Gimcheon, Gyeongsangbukdo, Korea; ᵇPrion Research Center (PRC), Colorado State University, Fort Collins, CO, USA</footnote>
<figure><img src="image_114.png" /></figure>

**Results:** Five randomly selected mice were sequentially culled on every 15 days from 30dpi to 120dpi during CWD infected TgElk mice reached terminal stage. Rough hair coats among clinical signs were showed from 90 dpi. $PrP^{CWD}$ in the brain in TgElk CWD was detectable persistently from early stages (30dpi), and in the kidney $PrP^{CWD}$ was also detectable in clinical and terminal stages (90 dpi and 120dpi). $PrP^{CWD}$ in the urine in TgElk CWD reached the highest levels at 120dpi. NaPTA/RT-QUIC was applied to measure $PrP^{CWD}$ in urine samples collected on every 15 days from 30dpi to 120dpi when CWD infected TgElk mice reached terminal stage. $PrP^{CWD}$ in the urine in TgElk CWD reached the highest levels at 90dpi. $PrP^{CWD}$ was also detectable in late and terminal stages (120dpi).

**Conclusions:** We demonstrate that CWD prions can be detected by RT-QUIC or NaPTA/RT-QUIC in the brain, kidney and urine of TgElk mice at the early and terminal stages of disease. Based on these data, we suggest that $PrP^{CWD}$ is excreted into only urine until 90 dpi and then slowly accumulated in kidney. Our results can be used in designing future study of CWD pathogenesis in TgElk mice.

## References

[1] Henderson DM et al., Rapid antemortem detection of CWD prions in deer saliva. PLOS one. 2013:e74377 1-12

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

## 180. Cell assay detection of chronic wasting disease (CWD) prion infectivity

Su Bi Ahnª, Hyo-Jin Kimª, Jifeng Bianᵇ, In-Soon Rohª, Hae-Eun Kangª, Hyun-Joo Sohnª

CONTACT Hyun-Joo Sohn

### ABSTRACT

Chronic wasting disease (CWD) is a prion disease affecting the animals in family Cervidae. Prions are the infectious agents that cause prion disease. Mouse prion bioassay is the most commonly used method for determine prion infectivity. Application of more efficient measurement method was required as several cases of CWD have been reported in South Korea. In this study, we adapted the modified cervid prion cell assay (CPCA) using $Elk21^-$ cells. The susceptible

$Elk21^-$ cells were exposed to various CWD brain homogenates, ranging from $10^{-2}$ to $10^{-5}$. The CPCA titers of elk CWD brain homogenates were $10^{5.8}$ [2001 (E190Y+229Y)] and $10^{6.5}$ [16FC050] units/g of brain, respectively. We also found the CWD prion titer of red and sika deer brain inocula in 2016 CWD cases, producing CPCA titers of $10^{5.8}$ and $10^{5.4}$ units/g of brain. These results were confirmed by western blotting. Comparing mouse titers of mouse ic LD50/g of cervid brain with CPCA titers, sensitivity of the cell-based measurement was similar to that of the mouse bioassay. Also, CPCA titers does not depend on CWD species, relate with mouse bioassay survival time. Consequently, our results demonstrate that CWD prion infectivity can be efficiently and quantitatively detected by cell based titration of CWD.

## 181. Application of the TSE-infected cell and RT-QUIC assay to discover novel natural products

Hyo-Jin Kim, Su Bi Ahn, In-Soon Roh, Kyung-Je Park, Hae-Eun Kang, Hyun-Joo Sohn

Foreign Animal Disease Division, Animal and Plant Quarantine Agency, Gimcheon, Gyeongsangbukdo, Korea

CONTACT Hyun-Joo Sohn

### ABSTRACT

Conformational conversion of the normal prion protein ($PrP^C$) into an infectious $PrP^{Sc}$ causes pathogenesis in prion diseases or transmissible spongiform encephalopathies (TSEs). TSE-infected cell lines have been efficiently used to study this conversion process, as well as to screen for potentially effective anti-prion compounds. We employed fragment molecular orbital (FMO)-based virtual screening and BSE-infected cell-based assay system to discover two natural products with antiprion activity exhibited good binding interactions, with hotspot residues within the $PrP^C$ binding site, and effectively reduced $PrP^{Sc}$ levels in a standard scrapie cell assay (SSCA). We also investigated the prion inhibition ability of two novel natural products in Elk $21^+$ (CWD) or SMB (Scrapie) cells. Cytotoxicity tests showed no significant cell death in either BNP-03 or BNP-08 treatment. Elk $21^+$ and SMB cells were subjected to two effective natural products for reducing $PrP^{Sc}$, BNP-03, and BNP-08. Efficiency of $PrP^{Sc}$ clearance for two natural products was estimated by evaluating the signal sensitivity for $PrP^{Sc}$ on SSCA, WB, and EIA. Antiprion activity of the compounds was detected by RT-QUIC assay seeded with TgElk CWD (E190Y +229Y). These natural products showed the inhibitory effects in the TSE-infected cells. Further experiments in

<footnote>ªForeign Animal Disease Division, Animal and Plant Quarantine Agency, Gimcheon, Gyeongsangbukdo, Korea; ᵇPrion Research Center (PRC), Colorado State University, Fort Collins, CO, USA</footnote>
<figure><img src="image_115.png" /></figure>

animal models will be necessary to confirm the effect of
two novel natural products *in vivo*.

182. Species and sex differences in contact during
the breeding season may explain variation in
Chronic Wasting Disease prevalence

Kelsey Saboraki and Susan Lingle

Department of Biology, University of Winnipeg

CONTACT Kelsey Saboraki ksaboraki@gmail.com

ABSTRACT

**Background:** Chronic Wasting Disease (CWD) is a transmissible and fatal neurodegenerative prion disease that threatens wild populations of ungulates. Prevalence of CWD is higher in mule deer than in white-tailed deer and in males than in females of both species. Prions are spread through direct and indirect contact with saliva, urine, faeces and to an unknown extent, glandular secretions. Species and sex differences in rates of direct contact with infected individuals or indirect contact with environmental contaminants may result in species and sex differences in pathogen transmission and disease prevalence. Social behaviours and the potential for pathogen transmission change seasonally, and interactions during the breeding season may explain differences in prevalence. We therefore tested the hypothesis that species and sex differences in behaviour during the breeding season underlie species and sex differences in CWD prevalence in mule and white-tailed deer.

**Methods:** We observed behavioural interactions between deer during the breeding season on an open grassland site, the McIntyre Ranch, in southern Alberta to assess whether species and sex differences in direct physical contact (deer to deer) and indirect contact (deer to environment to deer) correspond to the higher prevalence of CWD in mule deer and in males. We observed 86 deer (21 mule deer females, 21 mule deer males, 20 white-tailed females, and 24 white-tailed males) and recorded the occurrence and duration of deer-deer and deer-environment-deer contact and the number of partners with which deer engaged in contact with over time.

**Results and Discussion:** We found that males exhibit sex-specific behaviours, such as marking vegetation and flehmen, that have the potential to elevate their risk of prion transmission relative to females. During courtship interactions, between sex contact was more common in mule deer than in white-tailed

deer, which could explain species differences in prevalence. As females are in estrous for only 1–2 days while a successful male engages in high-risk courtship throughout the rut, these results show that males of both species should have a higher cumulative potential for prion transmission throughout the breeding season. By collecting empirical data on the fine-scale behaviour of sympatric mule deer and white-tailed deer during the breeding season, we identified mechanisms that may be responsible for the higher prevalence of CWD in mule deer and in males.

**KEYWORDS:** Chronic wasting disease; mule deer; white-tailed deer; behaviour; transmission

183. Morphometric analysis of PrP deposition in
two models of prion diseases

Pawel P. Liberski<sup>a</sup>, Agata Gajos<sup>b</sup>, Beata Sikorska<sup>a</sup>
and Janusz Moryś<sup>c</sup>

CONTACT Pawel P. Liberski

ABSTRACT

The Echigo-1 strain of Creutzfeldt-Jakob disease (CJD) was isolated from a case of a 33-years-old female with a panencephalopathic type of CJD. The Fujisaki (Fukuoka 1) was isolated from a case of Gerstmann-Straussler-Scheinker disease (GSS)

**Material and Methods:** The morphometrical analysis was done using the Zen Blue v. 2.6 and its automated analysis module for calculation of percentage of total area of all immunopositive objects in relation to the area of the measurement frame as well as the mean intensity of grey values of an analysed region and the pathological changes.

**Results:** The significant differences in the distribution of pathological changes were observed in the studied two groups of animals. In the Echigo-1 animals the most intense changes were observed in the lateral dorsal nucleus (49.8% of studied area was occupied by the pathological protein) and ventrolateral nucleus (20.3%) of the thalamus, auditory (37.8%) and somatosensory (32.9%) cortices and the hilus (22.4%), and CA2 (28.6%) sector of the hippocampus. The smallest changes were present in the dentate gyrus (1.4%) and putamen (5.1%). The highest reaction of the microglia was observed in the somatosensory cortex, lateral dorsal nucleus, and in the hippocampal sectors. Very

<footnote><sup>a</sup>Laboratory of Electron Microscopy and Neuropathology, Department of Molecular Pathology and Neuropathology, Chair of Oncology, Medical University Lodz, Poland; <sup>b</sup>Department of Extrapyramidal Diseases, Chair of Rehabilitation, Medical University of Lodz, Lodz, Poland; <sup>c</sup>Department of Anatomy and Neurobiology, Medical University of Gdansk, Gdansk, Poland</footnote>
<figure><img src="image_115.png" /></figure>

animal models will be necessary to confirm the effect of
two novel natural products *in vivo*.

182. Species and sex differences in contact during
the breeding season may explain variation in
Chronic Wasting Disease prevalence

Kelsey Saboraki and Susan Lingle

Department of Biology, University of Winnipeg

CONTACT Kelsey Saboraki ksaboraki@gmail.com

ABSTRACT

**Background:** Chronic Wasting Disease (CWD) is a transmissible and fatal neurodegenerative prion disease that threatens wild populations of ungulates. Prevalence of CWD is higher in mule deer than in white-tailed deer and in males than in females of both species. Prions are spread through direct and indirect contact with saliva, urine, faeces and to an unknown extent, glandular secretions. Species and sex differences in rates of direct contact with infected individuals or indirect contact with environmental contaminants may result in species and sex differences in pathogen transmission and disease prevalence. Social behaviours and the potential for pathogen transmission change seasonally, and interactions during the breeding season may explain differences in prevalence. We therefore tested the hypothesis that species and sex differences in behaviour during the breeding season underlie species and sex differences in CWD prevalence in mule and white-tailed deer.

**Methods:** We observed behavioural interactions between deer during the breeding season on an open grassland site, the McIntyre Ranch, in southern Alberta to assess whether species and sex differences in direct physical contact (deer to deer) and indirect contact (deer to environment to deer) correspond to the higher prevalence of CWD in mule deer and in males. We observed 86 deer (21 mule deer females, 21 mule deer males, 20 white-tailed females, and 24 white-tailed males) and recorded the occurrence and duration of deer-deer and deer-environment-deer contact and the number of partners with which deer engaged in contact with over time.

**Results and Discussion:** We found that males exhibit sex-specific behaviours, such as marking vegetation and flehmen, that have the potential to elevate their risk of prion transmission relative to females. During courtship interactions, between sex contact was more common in mule deer than in white-tailed

deer, which could explain species differences in prevalence. As females are in estrous for only 1–2 days while a successful male engages in high-risk courtship throughout the rut, these results show that males of both species should have a higher cumulative potential for prion transmission throughout the breeding season. By collecting empirical data on the fine-scale behaviour of sympatric mule deer and white-tailed deer during the breeding season, we identified mechanisms that may be responsible for the higher prevalence of CWD in mule deer and in males.

**KEYWORDS:** Chronic wasting disease; mule deer; white-tailed deer; behaviour; transmission

183. Morphometric analysis of PrP deposition in
two models of prion diseases

Pawel P. Liberski<sup>a</sup>, Agata Gajos<sup>b</sup>, Beata Sikorska<sup>a</sup>
and Janusz Moryś<sup>c</sup>

CONTACT Pawel P. Liberski

ABSTRACT

The Echigo-1 strain of Creutzfeldt-Jakob disease (CJD) was isolated from a case of a 33-years-old female with a panencephalopathic type of CJD. The Fujisaki (Fukuoka 1) was isolated from a case of Gerstmann-Straussler-Scheinker disease (GSS)

**Material and Methods:** The morphometrical analysis was done using the Zen Blue v. 2.6 and its automated analysis module for calculation of percentage of total area of all immunopositive objects in relation to the area of the measurement frame as well as the mean intensity of grey values of an analysed region and the pathological changes.

**Results:** The significant differences in the distribution of pathological changes were observed in the studied two groups of animals. In the Echigo-1 animals the most intense changes were observed in the lateral dorsal nucleus (49.8% of studied area was occupied by the pathological protein) and ventrolateral nucleus (20.3%) of the thalamus, auditory (37.8%) and somatosensory (32.9%) cortices and the hilus (22.4%), and CA2 (28.6%) sector of the hippocampus. The smallest changes were present in the dentate gyrus (1.4%) and putamen (5.1%). The highest reaction of the microglia was observed in the somatosensory cortex, lateral dorsal nucleus, and in the hippocampal sectors. Very

<footnote><sup>a</sup>Laboratory of Electron Microscopy and Neuropathology, Department of Molecular Pathology and Neuropathology, Chair of Oncology, Medical University Lodz, Poland; <sup>b</sup>Department of Extrapyramidal Diseases, Chair of Rehabilitation, Medical University of Lodz, Lodz, Poland; <sup>c</sup>Department of Anatomy and Neurobiology, Medical University of Gdansk, Gdansk, Poland</footnote>
<figure><img src="image_116.png" /></figure>

weak reaction of the microglia was present in the dentate hilus, putamen, and ventrolateral thalamus.

In the second group of animals the most dramatic changes were observed in the thalamus (ventrolateral nucleus – 56.2%; lateral dorsal nucleus – 50.3%, and lateral posterior nucleus – 40.7%) as well as in the hippocampus (CA1 – 48.1%, CA3 – 40.3%, and CA2 – 35.6). The moderate reaction was present the somatosensory and auditory cortex, basolateral amygdala, and putamen. The weak reaction was observed in the dentate gyrus and endopiriform nucleus. The intensive reaction of microglia was present in the somatosensory cortex, in the thalamus and basolateral amygdala. The small reactivity of the microglia was observed in the dentate gyrus and its hilus as well as in the CA1 sector. The microglia reactivity was almost twice bigger in the present in the animals injected by the Fujisaki strain than Echigo-1, also the intensity of immunoreactivity for prion protein distribution was biggest in the first group of animals.

## Funding

PPL and BS are supported by National Science Centre Poland, grant number UMO-2015/19/B/NZ4/03234

## 184. Complex pathways for native folding and misfolding of SOD1 observed directly by single-molecule force spectroscopy

Supratik Sen Mojumdar, Craig R. Garen and Michael T. Woodside

University of Alberta

CONTACT Michael T. Woodside

## ABSTRACT

Superoxide dismutase 1 (SOD1) is a β-barrel dimer that undergoes prion-like misfolding in the context of ALS, but its misfolding mechanism remains unclear. We studied the folding of SOD1 monomers and dimers by using laser tweezers to unfold and refold single molecules held under mechanical tension. Monomers were found to fold via numerous intermediate states, in contrast to the two-state folding deduced from ensemble studies. A novel pathway analysis indicated that these intermediates involve the addition of individual β-strands to the native structure. Several misfolded states were also observed, branching off from the native pathway after the formation of a stable native-like core [Sen Mojumdar et al., Nat. Commun. 2017]. In dimers, the interface between domains changed the folding noticeably, in

several ways. First, the number of intermediates was lower than expected for independent monomers, indicating increased cooperativity. Second, the dimer interface was found to modulate the folding pathways by changing the relative stability of the monomer segments close to the interface. Third, the dimer misfolded only half as much as expected from the monomer misfolding rate, suggesting the interface helps protect against misfolding, in part by reducing the prevalence of partially-folded intermediates that lead to misfolding. Finally, inserting the point mutation G85R, which is associated with familial ALS, vastly increased the rate of misfolding.

## 185. Probing cortico-cortical and hippocampal-cortical interactions in a second-generation mouse model of Alzheimer's disease

Surjeet Singh, Jogender Mehla, Robert J. Sutherland and Majid Mohajerani

Department of Neuroscience, Canadian Center for Behavioral Neuroscience, University of Lethbridge, Alberta, Canada

CONTACT Surjeet Singh

## ABSTRACT

It is known that Alzheimer's disease (AD) is associated with defects of synaptic connectivity. Such defects may not be restricted to local neuronal interactions but may extend to long-range brain activities, such as slow-wave oscillations that are particularly prominent during non-rapid eye movement (non-REM) sleep and are important for integration of information across distant brain regions involved in memory consolidation. Using a newly characterized knock-in mouse model of AD (n = 8, 12 months old) that has humanized amyloid beta (Aβ) sequence knocked-in to the murine amyloid precursor protein gene along with the Swedish, Arctic, and Beyreuther/Iberian mutations (APP NL-G-F), and wide-field optical imaging of cortical voltage responses (VSDI) combined with local field potential (LFP) recording from CA1, we investigated that how Aβ deposition in these mouse model impacts cortical functional connectivity and how different cortical regions interact with hippocampus during slow wave sleep. We found that Aβ deposition in knock-in mouse model of AD (APP NL-G-F) impairs cortical functional connectivity and intrinsic hippocampal circuit that generates SWR. Coordination of cortical up-states and hippocampal sharp-wave ripples (HPC-SWR) during slow wave sleep is believed to play a major role in the consolidation of recently acquired memories. It was observed that HPC-SWR show strong correlation with
<figure><img src="image_116.png" /></figure>

weak reaction of the microglia was present in the dentate hilus, putamen, and ventrolateral thalamus.

In the second group of animals the most dramatic changes were observed in the thalamus (ventrolateral nucleus – 56.2%; lateral dorsal nucleus – 50.3%, and lateral posterior nucleus – 40.7%) as well as in the hippocampus (CA1 – 48.1%, CA3 – 40.3%, and CA2 – 35.6). The moderate reaction was present the somatosensory and auditory cortex, basolateral amygdala, and putamen. The weak reaction was observed in the dentate gyrus and endopiriform nucleus. The intensive reaction of microglia was present in the somatosensory cortex, in the thalamus and basolateral amygdala. The small reactivity of the microglia was observed in the dentate gyrus and its hilus as well as in the CA1 sector. The microglia reactivity was almost twice bigger in the present in the animals injected by the Fujisaki strain than Echigo-1, also the intensity of immunoreactivity for prion protein distribution was biggest in the first group of animals.

## Funding

PPL and BS are supported by National Science Centre Poland, grant number UMO-2015/19/B/NZ4/03234

## 184. Complex pathways for native folding and misfolding of SOD1 observed directly by single-molecule force spectroscopy

Supratik Sen Mojumdar, Craig R. Garen and Michael T. Woodside

University of Alberta

CONTACT Michael T. Woodside

## ABSTRACT

Superoxide dismutase 1 (SOD1) is a β-barrel dimer that undergoes prion-like misfolding in the context of ALS, but its misfolding mechanism remains unclear. We studied the folding of SOD1 monomers and dimers by using laser tweezers to unfold and refold single molecules held under mechanical tension. Monomers were found to fold via numerous intermediate states, in contrast to the two-state folding deduced from ensemble studies. A novel pathway analysis indicated that these intermediates involve the addition of individual β-strands to the native structure. Several misfolded states were also observed, branching off from the native pathway after the formation of a stable native-like core [Sen Mojumdar et al., Nat. Commun. 2017]. In dimers, the interface between domains changed the folding noticeably, in

several ways. First, the number of intermediates was lower than expected for independent monomers, indicating increased cooperativity. Second, the dimer interface was found to modulate the folding pathways by changing the relative stability of the monomer segments close to the interface. Third, the dimer misfolded only half as much as expected from the monomer misfolding rate, suggesting the interface helps protect against misfolding, in part by reducing the prevalence of partially-folded intermediates that lead to misfolding. Finally, inserting the point mutation G85R, which is associated with familial ALS, vastly increased the rate of misfolding.

## 185. Probing cortico-cortical and hippocampal-cortical interactions in a second-generation mouse model of Alzheimer's disease

Surjeet Singh, Jogender Mehla, Robert J. Sutherland and Majid Mohajerani

Department of Neuroscience, Canadian Center for Behavioral Neuroscience, University of Lethbridge, Alberta, Canada

CONTACT Surjeet Singh

## ABSTRACT

It is known that Alzheimer's disease (AD) is associated with defects of synaptic connectivity. Such defects may not be restricted to local neuronal interactions but may extend to long-range brain activities, such as slow-wave oscillations that are particularly prominent during non-rapid eye movement (non-REM) sleep and are important for integration of information across distant brain regions involved in memory consolidation. Using a newly characterized knock-in mouse model of AD (n = 8, 12 months old) that has humanized amyloid beta (Aβ) sequence knocked-in to the murine amyloid precursor protein gene along with the Swedish, Arctic, and Beyreuther/Iberian mutations (APP NL-G-F), and wide-field optical imaging of cortical voltage responses (VSDI) combined with local field potential (LFP) recording from CA1, we investigated that how Aβ deposition in these mouse model impacts cortical functional connectivity and how different cortical regions interact with hippocampus during slow wave sleep. We found that Aβ deposition in knock-in mouse model of AD (APP NL-G-F) impairs cortical functional connectivity and intrinsic hippocampal circuit that generates SWR. Coordination of cortical up-states and hippocampal sharp-wave ripples (HPC-SWR) during slow wave sleep is believed to play a major role in the consolidation of recently acquired memories. It was observed that HPC-SWR show strong correlation with
cortical activation in retrosplenial cortex (RSC). However, the magnitude of ripple power and cortical activation in RSC is reduced in AD mice as compared littermate controls, suggesting impairment in hippocampal-cortical interaction during slow wave sleep. We further analysed cortical VSDI data from 19 region of interests (ROIs) with two nonlinear methods: entropy (En) and auto mutual information (AMI). En quantifies regularity in data, while AMI detects linear and nonlinear dependencies in time series. We observed that En was lower in AD mice, and AMI decreased more slowly with time delays in AD as compared to control mice, for both En and AMI significant differences were observed in occipital, somatosensory and motor cortex (p <0.05). These results provide new insights into brain dysfunction associated with Aß deposition in APP NL-G-F mouse model of AD.

**186. Serial detection of hematogenous prions in CWD-infected deer**

Amy V. Nalls, Erin E. McNulty, Nathaniel D. Denkers, Edward A. Hoover and Candace K. Mathiason

Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO, USA

CONTACT Amy V. Nalls amy.nalls@colostate.edu

**ABSTRACT**

Blood contains the infectious agent associated with prion disease affecting several mammalian species, including humans, cervids, sheep, and cattle. It has been confirmed that sufficient prion agent is present in the blood of both symptomatic and asymptomatic carriers to initiate the amyloid templating and accumulation process that results in this fatal neurodegenerative disease. Yet, to date, the ability to detect blood-borne prions by *in vitro* methods remains difficult.

We have capitalized on blood samples collected from longitudinal chronic wasting disease (CWD) studies in the native white-tailed deer host to examine hematogenous prion load in blood collected minutes, days, weeks and months post exposure. Our work has focused on refinement of the amplification methods RT-QuIC and PMCA. We demonstrate enhanced *in vitro* detection of amyloid seeding activity (prions) in blood cell fractions harvested from deer orally-exposed to 300 ng CWD positive brain or saliva.

These findings permit assessment of the role hematogenous prions play in the pathogenesis of CWD and provide tools to assess the same for prion diseases of other mammalian species.

**187. Impact of new Creutzfeldt-Jakob disease mouse models in prion research**

Juan Maria Torresª, Juan Carlos Espinosaª, Olivier Andreolettiᵇ, Isidre Ferrerᶜ, Inga Zerrᵈ, Franc Llorensᶜ,ᵈ

ªCentro de Investigación en Sanidad Animal-INIA, Madrid, Spain;
ᵇFrench National Institute for Agricultural Research (INRA)-ENVT UMR 1225, Toulouse, France; ᵇBellvitge Biomedical Research Institute, Barcelona, Spain and Network Center for Biomedical Research of Neurodegenerative Diseases, Institute Carlos III, Ministry of Health, Spain; ᵈDepartment of Neurology, University Medical School, Göttingen, Germany

CONTACT Franc Llorens franc.llorens@gmail.com

**ABSTRACT**

Studying animal prion diseases models is an appealing approach to dissect the temporal-dependent physiopathology of human prion diseases. In the recent years, new mouse models have been developed based on the genetic deletion of the endogenous mouse prion protein gene (PrnP 0/0) and the overexpression of the human prion protein (PRNP). After their inoculation with human brain homogenates, the animals develop a prion pathology, which highly recapitulates the neuropathological and biochemical features of human prion diseases in a subtype-dependent manner.

These humanized mouse models have shown to be crucial for the understanding of the genetic susceptibility to prions, the transmissibility properties of different prion subtypes and the zoonotic potential of prion strains. However, the precise molecular and signalling features occurring during the progression of disease pathology in these models have not been yet scrutinized in detail.

We used the tg340-MM mouse model (transgenic mice overexpressing the *PRNP* with Met/Met at its codon 129 inoculated with sCJD MM1 brain homogenate) as a model of sCJD subtype MM1 in order to gain insight into the molecular mechanism driving to human prion pathogenesis. Targeted and screening studies were carried out in a temporal and regional-dependent fashion and main outcomes were systematically compared to those obtained in sCJD MM1 post-mortem cases.

In the tg340-MM mice, we detected temporal and regional dependent alterations in neuro-inflammatory profiles, miRNA, gene expression signatures, prion protein deposition patterns and pathogenic-associated cell signalling pathways that were highly similar than those detected in sCJD MM1 cases. Additionally, the study of the tg340-MM mice allowed us to profile the molecular signatures at preclinical and early-clinical stages of the disease.

Altogether, we proved the usefulness of new transgenic humanized CJD models as a valid tool for the
cortical activation in retrosplenial cortex (RSC). However, the magnitude of ripple power and cortical activation in RSC is reduced in AD mice as compared littermate controls, suggesting impairment in hippocampal-cortical interaction during slow wave sleep. We further analysed cortical VSDI data from 19 region of interests (ROIs) with two nonlinear methods: entropy (En) and auto mutual information (AMI). En quantifies regularity in data, while AMI detects linear and nonlinear dependencies in time series. We observed that En was lower in AD mice, and AMI decreased more slowly with time delays in AD as compared to control mice, for both En and AMI significant differences were observed in occipital, somatosensory and motor cortex (p <0.05). These results provide new insights into brain dysfunction associated with Aß deposition in APP NL-G-F mouse model of AD.

**186. Serial detection of hematogenous prions in CWD-infected deer**

Amy V. Nalls, Erin E. McNulty, Nathaniel D. Denkers, Edward A. Hoover and Candace K. Mathiason

Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO, USA

CONTACT Amy V. Nalls amy.nalls@colostate.edu

ABSTRACT

Blood contains the infectious agent associated with prion disease affecting several mammalian species, including humans, cervids, sheep, and cattle. It has been confirmed that sufficient prion agent is present in the blood of both symptomatic and asymptomatic carriers to initiate the amyloid templating and accumulation process that results in this fatal neurodegenerative disease. Yet, to date, the ability to detect blood-borne prions by *in vitro* methods remains difficult.

We have capitalized on blood samples collected from longitudinal chronic wasting disease (CWD) studies in the native white-tailed deer host to examine hematogenous prion load in blood collected minutes, days, weeks and months post exposure. Our work has focused on refinement of the amplification methods RT-QuIC and PMCA. We demonstrate enhanced *in vitro* detection of amyloid seeding activity (prions) in blood cell fractions harvested from deer orally-exposed to 300 ng CWD positive brain or saliva.

These findings permit assessment of the role hematogenous prions play in the pathogenesis of CWD and provide tools to assess the same for prion diseases of other mammalian species.

**187. Impact of new Creutzfeldt-Jakob disease mouse models in prion research**

Juan Maria Torresª, Juan Carlos Espinosaª, Olivier Andreolettiᵇ, Isidre Ferrerᶜ, Inga Zerrᵈ, Franc Llorensᶜ,ᵈ

ªCentro de Investigación en Sanidad Animal-INIA, Madrid, Spain;
ᵇFrench National Institute for Agricultural Research (INRA)-ENVT UMR 1225, Toulouse, France; ᵇBellvitge Biomedical Research Institute, Barcelona, Spain and Network Center for Biomedical Research of Neurodegenerative Diseases, Institute Carlos III, Ministry of Health, Spain; ᵈDepartment of Neurology, University Medical School, Göttingen, Germany

CONTACT Franc Llorens franc.llorens@gmail.com

ABSTRACT

Studying animal prion diseases models is an appealing approach to dissect the temporal-dependent physiopathology of human prion diseases. In the recent years, new mouse models have been developed based on the genetic deletion of the endogenous mouse prion protein gene (PrnP 0/0) and the overexpression of the human prion protein (PRNP). After their inoculation with human brain homogenates, the animals develop a prion pathology, which highly recapitulates the neuropathological and biochemical features of human prion diseases in a subtype-dependent manner.

These humanized mouse models have shown to be crucial for the understanding of the genetic susceptibility to prions, the transmissibility properties of different prion subtypes and the zoonotic potential of prion strains. However, the precise molecular and signalling features occurring during the progression of disease pathology in these models have not been yet scrutinized in detail.

We used the tg340-MM mouse model (transgenic mice overexpressing the *PRNP* with Met/Met at its codon 129 inoculated with sCJD MM1 brain homogenate) as a model of sCJD subtype MM1 in order to gain insight into the molecular mechanism driving to human prion pathogenesis. Targeted and screening studies were carried out in a temporal and regional-dependent fashion and main outcomes were systematically compared to those obtained in sCJD MM1 post-mortem cases.

In the tg340-MM mice, we detected temporal and regional dependent alterations in neuro-inflammatory profiles, miRNA, gene expression signatures, prion protein deposition patterns and pathogenic-associated cell signalling pathways that were highly similar than those detected in sCJD MM1 cases. Additionally, the study of the tg340-MM mice allowed us to profile the molecular signatures at preclinical and early-clinical stages of the disease.

Altogether, we proved the usefulness of new transgenic humanized CJD models as a valid tool for the
<figure><img src="image_117.png" /></figure>

study of the molecular features associated with human
prion diseases.

**188. Full atomistic model of PrPSc structure and conversion**

Giovanni Spagnolli<sup>a</sup>, Marta Rigoli<sup>a</sup>, Simone Orioli<sup>a,b</sup>, Alejandro M Sevillano<sup>c</sup>, Pietro Faccioli<sup>a,b</sup>, Holger Wille<sup>d</sup>, Emiliano Biasini<sup>a</sup>, Jesus R Requena<sup>e</sup>

CONTACT Giovanni Spagnolli giovanni.spagnolli@unitn.it

ABSTRACT

Since their discovery, prions have stood as enigmatic
agents that defy the classical concept of genetic inheri-
tance due to the capacity of propagating their conforma-
tionally encoded information in absence of nucleic acid
[1]. However, after more than 30 years, the high resolution
structure of PrPSc, required to understand the molecular
features of prion infectivity, has remained elusive [2].
Different PrPSc models have been proposed but they are
now inconsistent with recent results [2]. In this work,
using molecular modelling techniques, we constructed
an all-atom model of mouse PrPSc, derived from the
integration of a wide array of experimental constraints,
such as: (i) cryo-EM and X-ray fiber-diffraction studies,
which showed that the fold of a mouse infectious PrP is
compatible with a 4-rung-β-solenoid (4RβS) architecture
[3,4]; (ii) circular dichroism and FTIR spectroscopy,
which ruled out the presence of α-helices [5]; (iii) mass
spectrometry analyses, indicating the presence of an intact
disulphide bond between residues C178 and C213 [6], as
well as mapping Proteinase K sensitive residues, which
reflect amino acids likely excluded from the resistant core
of the protein [7]; and (iv) the possibility of accommodat-
ing complex glycans at positions N180 and N196 [8]. The
stability of this new PrP<sub>Sc</sub> model, assessed by Molecular
Dynamics simulations, was found to be comparable to
that of the prion forming domain of Het-s, a naturally-
occurring β-solenoid protein. Finally, we coupled the
information of the 4RβS structure with Ratchet and
Pawl Molecular Dynamics [9], to perform for the first
time a simulation of the conformational transition from
PrPC to PrP<sub>Sc</sub>. The result is a transition pathway with
atomistic resolution in which the C-terminal rung of the
solenoid acts as a primary conversion surface for PrPC
unstructured N-terminus, followed by a cascade of con-
formational changes where each newly formed rung tem-
plates the formation of the following one, ultimately

leading to the complete conversion of PrPC into PrP<sub>Sc</sub>.
This structure is, to the best of our knowledge, the first
physically plausible PrP<sub>Sc</sub> model, and represents a unique
workbench for interpreting future structural data or avail-
able biological evidence, such as the effects of variations in
the *PRNP* gene favoring or disfavoring prion propagation.
Furthermore, it allowed us to reconstruct how the infor-
mation encoded into the conformation of a protein could
be propagated in a directional fashion: a concept under-
lying the infectious nature of prions.

**KEYWORDS:** PrP<sub>Sc</sub> Structure; Prion Propagation; Molecular Modelling; Molecular Dynamics

References

[1] Prusiner SB. Science. 1982;216(4542):136-144.

[2] Wille H, Requena JR. Pathogens. 2018;7(1):20.

[3] Vazquez Fernandez E, et al. PLoS Pathog. 2016;12(9):e1005835.

[4] Wille H, et al. Proc Natl Acad Sci USA. 2009;106(40):16990-16995.

[5] Requena JR, Wille H. Prion. 2014;8(1):60-66.

[6] Welker E, et al. J Biol Chem. 2002;277(36):33477-33481.

[7] Vazquez Fernandez E, et al. PLoS Pathog. 2012;14(1):e1006797.

[8] Baskakov IV, Katorcha E. Front Neurosci. 2016;10:358.

[9] Tiana G, Camilloni C. J Chem Phys. 2012;137(23):235101.

189. Prion substrain specific tropism for the
spleen: role of PrPC expression levels

Fabienne Reine<sup>a</sup>, Laetitia Herzog<sup>a</sup>, Philippe Tixador<sup>a</sup>, Thanh-Lan La<sup>a</sup>, Johann Castille<sup>b</sup>, Bruno Passet<sup>b</sup>, Human Rezaei<sup>a</sup>, Olivier Andréoletti<sup>c</sup>, Jean-Luc Vilotte<sup>b</sup>, Hubert Laude<sup>a</sup> and Vincent Béringue<sup>a</sup>

CONTACT Vincent Béringue vincent.beringue@inra.fr

ABSTRACT

Prions are proteinaceous pathogens formed from mis-
folded conformers (PrPSc) of the host encoded cellular
prion protein (PrPC). Prions replicate by templating the
conversion and polymerization of PrPC by an autocata-
lytic process. Multiple strains of prions are recognized
phenotypically within the same host species. Strains are
conformational variants of PrPSc at the level of the ter-
tiary and the quaternary structure. In infected hosts, prion
strains exhibit a stereotyped biological phenotype, that
can include specific tropism for the lymphoid tissue.

<footnote><sup>a</sup>University of Trento, Italy; <sup>b</sup>Italian Institute for Nuclear Physics (INFN) - TIFPA, Italy; <sup>c</sup>University of California San Diego, USA; <sup>d</sup>University of Alberta, Canada; <sup>e</sup>University of Santiago de Compostela, Spain</footnote>

<footnote><sup>a</sup>VIM, INRA, Université Paris-Saclay, Jouy-en-Josas, France; <sup>b</sup>GABI, INRA, AgroParisTech, Université Paris-Saclay, Jouy-en-Josas, France; <sup>c</sup>IHAP, INRA, ENVT, Toulouse, France</footnote>
<figure><img src="image_117.png" /></figure>

study of the molecular features associated with human
prion diseases.

## 188. Full atomistic model of PrPSc structure and conversion

Giovanni Spagnolliª, Marta Rigoliª, Simone Orioliª,ᵇ,
Alejandro M Sevillanoᶜ, Pietro Faccioliª,ᵇ, Holger
Willeᵈ, Emiliano Biasiniª, Jesus R Requenaᵉ

CONTACT Giovanni Spagnolli giovanni.spagnolli@unitn.it

## ABSTRACT

Since their discovery, prions have stood as enigmatic
agents that defy the classical concept of genetic inheri-
tance due to the capacity of propagating their conforma-
tionally encoded information in absence of nucleic acid
[1]. However, after more than 30 years, the high resolution
structure of PrPSc, required to understand the molecular
features of prion infectivity, has remained elusive [2].
Different PrPSc models have been proposed but they are
now inconsistent with recent results [2]. In this work,
using molecular modelling techniques, we constructed
an all-atom model of mouse PrPSc, derived from the
integration of a wide array of experimental constraints,
such as: (i) cryo-EM and X-ray fiber-diffraction studies,
which showed that the fold of a mouse infectious PrP is
compatible with a 4-rung-β-solenoid (4RβS) architecture
[3,4]; (ii) circular dichroism and FTIR spectroscopy,
which ruled out the presence of α-helices [5]; (iii) mass
spectrometry analyses, indicating the presence of an intact
disulphide bond between residues C178 and C213 [6], as
well as mapping Proteinase K sensitive residues, which
reflect amino acids likely excluded from the resistant core
of the protein [7]; and (iv) the possibility of accommodat-
ing complex glycans at positions N180 and N196 [8]. The
stability of this new PrPSc model, assessed by Molecular
Dynamics simulations, was found to be comparable to
that of the prion forming domain of Het-s, a naturally-
occurring β-solenoid protein. Finally, we coupled the
information of the 4RβS structure with Ratchet and
Pawl Molecular Dynamics [9], to perform for the first
time a simulation of the conformational transition from
PrPC to PrPSc. The result is a transition pathway with
atomistic resolution in which the C-terminal rung of the
solenoid acts as a primary conversion surface for PrPC
unstructured N-terminus, followed by a cascade of con-
formational changes where each newly formed rung tem-
plates the formation of the following one, ultimately

leading to the complete conversion of PrPC into PrP<sub>Sc</sub>. This structure is, to the best of our knowledge, the first physically plausible PrP<sub>Sc</sub> model, and represents a unique workbench for interpreting future structural data or available biological evidence, such as the effects of variations in the PRNP gene favoring or disfavoring prion propagation. Furthermore, it allowed us to reconstruct how the information encoded into the conformation of a protein could be propagated in a directional fashion: a concept underlying the infectious nature of prions.

**KEYWORDS:** PrP<sub>Sc</sub> Structure; Prion Propagation; Molecular Modelling; Molecular Dynamics

## References

[1] Prusiner SB. Science. 1982;216(4542):136-144.

[2] Wille H, Requena JR. Pathogens. 2018;7(1):20.

[3] Vazquez Fernandez E, et al. PLoS Pathog. 2016;12(9):e1005835.

[4] Wille H, et al. Proc Natl Acad Sci USA. 2009;106(40):16990-16995.

[5] Requena JR, Wille H. Prion. 2014;8(1):60-66.

[6] Welker E, et al. J Biol Chem. 2002;277(36):33477-33481.

[7] Vazquez Fernandez E, et al. PLoS Pathog. 2012;14(1):e1006797.

[8] Baskakov IV, Katorcha E. Front Neurosci. 2016;10:358.

[9] Tiana G, Camilloni C. J Chem Phys. 2012;137(23):235101.

## 189. Prion substrain specific tropism for the spleen: role of PrPC expression levels

Fabienne Reineª, Laetitia Herzogª, Philippe Tixadorª,
Thanh-Lan Laia, Johann Castilleb, Bruno Passetb,
Human Rezaeia, Olivier Andréoletti c, Jean-Luc
Vilotteb, Hubert Laudeª and Vincent Béringueª

CONTACT Vincent Béringue vincent.beringue@inra.fr

## ABSTRACT

Prions are proteinaceous pathogens formed from mis-
folded conformers (PrPSc) of the host encoded cellular
prion protein (PrPC). Prions replicate by templating the
conversion and polymerization of PrPC by an autocata-
lytic process. Multiple strains of prions are recognized
phenotypically within the same host species. Strains are
conformational variants of PrPSc at the level of the ter-
tiary and the quaternary structure. In infected hosts, prion
strains exhibit a stereotyped biological phenotype, that
can include specific tropism for the lymphoid tissue.

<footnote>ªUniversity of Trento, Italy; ᵇItalian Institute for Nuclear Physics (INFN) - TIFPA, Italy; ᶜUniversity of California San Diego, USA; ᵈUniversity of Alberta, Canada; ᵉUniversity of Santiago de Compostela, Spain</footnote>

<footnote>ªVIM, INRA, Université Paris-Saclay, Jouy-en-Josas, France; ᵇGABI, INRA, AgroParisTech, Université Paris-Saclay, Jouy-en-Josas, France; ᶜIHAP, INRA, ENVT, Toulouse, France</footnote>
Prion isolates and strains can be composed of several
substrain components. These substrains can propagate
in distinct tissues of the same infected individual (e.g.
brain and spleen), in both homotypic and heterotypic
PrP transmission contexts [1-3]. The reason for such
tissue selectivity is unclear. Previously, we demonstrated
that PrPC expression levels in the brain govern prion
substrain selection during homotypic PrP transmission
events [4]. Thus, transmission of natural sheep scrapie
isolates (termed LAN) to ovine PrP transgenic mice
expressing different levels of PrPC in the brain resulted
in the phenotypic expression of the dominant LAN sub-
strain in mice expressing near physiological PrPC levels,
whereas a minor substrain was preferentially selected on
high-expressors.

Considering that PrPC expression levels are markedly
decreased in the spleen compared to the brain in wild-type
and PrP transgenic mouse models1, we questioned
whether PrPC amount could drive prion substrain spleen-
selectivity. The outcome of LAN sheep scrapie isolates
transmission in the spleen from high-expressors correlated
with the replication rate dependency on PrPC amount,
with exclusive and prominent colonization of the spleen
by the substrain preferentially replicating on low-expres-
sors. After intraperitoneal inoculation of LAN isolates,
such early spleen colonization allowed the neuropatholo-
gical expression of this otherwise brain-unfavoured sub-
strain. In addition, a pair of strain variants resulting from
the adaptation of cortical MM2 CJD subtype to ovine high-
expressors, and exhibiting differing brain vs. spleen trop-
ism in these mice [3], showed opposite tropism in low-
expressors.

Collectively, our results support the view that PrPC
expression levels are instrumental in prion substrain
specific tropism for the lymphoid tissue.

References

[1] Beringue V. et al., Science. 2012;335:472-475.

[2] Beringue V. et al., PLoS One. 2008;3:e1419.

[3] Chapuis J. et al., Acta Neuropathol Commun. 2016;4:2-15.

[4] Le Dur A. et al., Nat Commun. 2017;8:14,170.

190. Independent amplification of co-infected long incubation period low conversion efficiency prion strains

Thomas E. Eckland, Ronald A. Shikiya and Jason C. Bartz

Department of Medical Microbiology and Immunology, School of
Medicine, Creighton University, Omaha, NE, USA

CONTACT Jason C. Bartz jbartz@creighton.edu

ABSTRACT

Prion diseases are caused by a misfolded isoform of the prion protein, PrP<sup>C</sup>. Prion strains are encoded by strain-specific conformations of PrP<sup>C</sup> and prions can interfere with each other when a long-incubation period strain inhibits the conversion of a short-incubation period strain. Prion strain interference influences the emergence of a strain from a mixture and prion strain dynamics. It is unknown, however, if two long-incubation period strains can interfere with each other. Here, we show that co-infection of animals with combinations of long-incubation period prion strains failed to identify evidence of strain interference. To exclude the possibility that this inability of strains to interfere *in vivo* was due to a failure to infect common populations of neurons we used protein misfolding cyclic amplification strain interference (PMCAsi). Consistent with the animal bioassay studies, PMCAsi indicated that both co-infecting strains were amplifying independently, suggesting that the lack of strain interference is not due to a failure to target the same cells but is an inherent property of the strains involved. Importantly PMCA reactions seeded with long incubation-period strains contained relatively higher levels of remaining PrP<sup>C</sup> compared to reactions seeded with a short-incubation period strain. Mechanistically we hypothesize that co-infection with prion strains with relatively low prion conversion efficiencies, the abundance of PrP<sup>C</sup> is not limiting *in vivo* or *in vitro*. Overall, this observation changes the paradigm of the interactions of prion strains and has implications for interspecies transmission and emergence of prion strains from a mixture.

191. Neutralization of TACE a -secretase by ROCK and PDK1 kinases contributes to neurodegeneration in prion diseases: loss-of-PrPC protective function involved

Mathéa Pietriª, Aurélie Alleaume-Butauxª, Juliette Ezpeletaª, Vincent Baudouinª, Zaira Arellano-Anayaª, Anne Baudryª, Stéphane Haikᵇ, Yannick Baillyᶜ, Jean-Michel Peyrinᵈ, Odile Kellermannª, Jean-Marie Launayᵉ and Benoit Schneiderª

CONTACT Benoit Schneider benoit.schneider@parisdescartes.fr

<footnote>ªUniversité Paris Descartes, Inserm UMR-S 1124, Paris, France;</footnote>

<footnote><sup>b</sup>INSERM UMR-S 1127, CNRS UMR 7225, Université Pierre-et-Marie</footnote>

<footnote>Curie, Paris, France; cCNRS UPR 3212, Strasbourg, France; dCNRS</footnote>

<footnote>UMR 8246, Université Pierre-et-Marie Curie, Paris, France; eINSERM</footnote>

<footnote>UMR-S 942, Hôpital Lariboisière, Paris, France & Pharma Research
Department, Hoffmann La Roche, Basel, Switzerland</footnote>
Prion isolates and strains can be composed of several
substrain components. These substrains can propagate
in distinct tissues of the same infected individual (e.g.
brain and spleen), in both homotypic and heterotypic
PrP transmission contexts [1-3]. The reason for such
tissue selectivity is unclear. Previously, we demonstrated
that PrPC expression levels in the brain govern prion
substrain selection during homotypic PrP transmission
events [4]. Thus, transmission of natural sheep scrapie
isolates (termed LAN) to ovine PrP transgenic mice
expressing different levels of PrPC in the brain resulted
in the phenotypic expression of the dominant LAN sub-
strain in mice expressing near physiological PrPC levels,
whereas a minor substrain was preferentially selected on
high-expressors.

Considering that PrPC expression levels are markedly
decreased in the spleen compared to the brain in wild-type
and PrP transgenic mouse models1, we questioned
whether PrPC amount could drive prion substrain spleen-
selectivity. The outcome of LAN sheep scrapie isolates
transmission in the spleen from high-expressors correlated
with the replication rate dependency on PrPC amount,
with exclusive and prominent colonization of the spleen
by the substrain preferentially replicating on low-expres-
sors. After intraperitoneal inoculation of LAN isolates,
such early spleen colonization allowed the neuropatholo-
gical expression of this otherwise brain-unfavoured sub-
strain. In addition, a pair of strain variants resulting from
the adaptation of cortical MM2 CJD subtype to ovine high-
expressors, and exhibiting differing brain vs. spleen trop-
ism in these mice [3], showed opposite tropism in low-
expressors.

Collectively, our results support the view that PrPC
expression levels are instrumental in prion substrain
specific tropism for the lymphoid tissue.

References

[1] Beringue V. et al., Science. 2012;335:472-475.

[2] Beringue V. et al., PLoS One. 2008;3:e1419.

[3] Chapuis J. et al., Acta Neuropathol Commun. 2016;4:2-15.

[4] Le Dur A. et al., Nat Commun. 2017;8:14,170.

190. Independent amplification of co-infected long incubation period low conversion efficiency prion strains

Thomas E. Eckland, Ronald A. Shikiya and Jason C. Bartz

Department of Medical Microbiology and Immunology, School of
Medicine, Creighton University, Omaha, NE, USA

CONTACT Jason C. Bartz jbartz@creighton.edu

ABSTRACT

Prion diseases are caused by a misfolded isoform of the
prion protein, PrP<sup>Sc</sup>. Prion strains are encoded by strain-
specific conformations of PrP<sup>Sc</sup> and prions can interfere
with each other when a long-incubation period strain
inhibits the conversion of a short-incubation period
strain. Prion strain interference influences the emer-
gence of a strain from a mixture and prion strain
dynamics. It is unknown, however, if two long-incuba-
tion period strains can interfere with each other. Here,
we show that co-infection of animals with combinations
of long-incubation period prion strains failed to identify
evidence of strain interference. To exclude the possibility
that this inability of strains to interfere *in vivo* was due to
a failure to infect common populations of neurons we
used protein misfolding cyclic amplification strain inter-
ference (PMCAsi). Consistent with the animal bioassay
studies, PMCAsi indicated that both co-infecting strains
were amplifying independently, suggesting that the lack
of strain interference is not due to a failure to target the
same cells but is an inherent property of the strains
involved. Importantly PMCA reactions seeded with
long incubation-period strains contained relatively
higher levels of remaining PrP<sup>C</sup> compared to reactions
seeded with a short-incubation period strain.
Mechanistically we hypothesize that co-infection with
prion strains with relatively low prion conversion effi-
ciencies, the abundance of PrP<sup>C</sup> is not limiting *in vivo* or
*in vitro*. Overall, this observation changes the paradigm
of the interactions of prion strains and has implications
for interspecies transmission and emergence of prion
strains from a mixture.

191. Neutralization of TACE a -secretase by ROCK and PDK1 kinases contributes to neurodegeneration in prion diseases: loss-of-PrPC protective function involved

Mathéa Pietriª, Aurélie Alleaume-Butauxª, Juliette Ezpeletaª, Vincent Baudouinª, Zaira Arellano-Anayaª, Anne Baudryª, Stéphane Haikᵇ, Yannick Baillyᶜ, Jean-Michel Peyrinᵈ, Odile Kellermannª, Jean-Marie Launayᵉ and Benoit Schneiderª

CONTACT Benoit Schneider benoit.schneider@parisdescartes.fr

<footnote>ªUniversité Paris Descartes, Inserm UMR-S 1124, Paris, France;</footnote>

<footnote><sup>b</sup>INSERM UMR-S 1127, CNRS UMR 7225, Université Pierre-et-Marie</footnote>

<footnote>Curie, Paris, France; cCNRS UPR 3212, Strasbourg, France; dCNRS</footnote>

<footnote>UMR 8246, Université Pierre-et-Marie Curie, Paris, France;</footnote>

<footnote>eINSERM
UMR-S 942, Hôpital Lariboisière, Paris, France & Pharma Research
Department, Hoffmann La Roche, Basel, Switzerland</footnote>
<figure><img src="image_118.png" /></figure>

## ABSTRACT

The α-secretase TACE exerts a protective role at the neuronal cell surface by catalysing the cleavage of several substrates, including (i) TNF α receptors (TNFRs), which confers physiological sensitivity to TNF α, and (ii) the cellular prion protein PrP<sup>C</sup>, which limits its conversion into pathogenic prions PrP<sup>Sc</sup>. We have assessed whether deregulation of TACE contributes to neurodegeneration in prion diseases.

Using the 1C11 neuronal cell line, primary cultures of neurons and mice infected with prions, we show TACE deficit at the surface of prion-infected cells. Mechanistically, PrP<sup>Sc</sup> promotes the internalization of TACE into caveolin-1-enriched vesicles, which diverts TACE activity away from TNFRs and PrP<sup>C</sup>. This renders neurons highly sensitive to TNF α-associated inflammation and amplifies the production of PrP<sup>Sc</sup> in prion-infected neurons. Whether the neuropathological effect of PrP<sup>Sc</sup>-induced TACE internalization also depends on the uncoupling of the α-secretase from other substrates is currently investigated.

We provide evidence that TACE internalization in prion-infected neurons originates from a loss-of-PrP<sup>C</sup> control of TACE localization at the cell surface upon PrP<sup>C</sup> conversion into PrP<sup>Sc</sup>. The silencing of PrP<sup>C</sup> in uninfected neurons or the conversion of PrP<sup>C</sup> into PrP<sup>Sc</sup> in prion-infected neurons both promote plasma membrane aggregation of β 1 integrins and a rise of β 1 integrin signalling. This leads to the downstream over-activation of the kinase ROCK that complexes with another kinase, PDK1, and overstimulates PDK1 activity. Overactivated PDK1 then triggers the displacement of TACE from the plasma membrane to caveolin 1-enriched vesicles. The inhibition of ROCK or PDK1 is sufficient to target TACE back to the plasma membrane where TACE recovers its protective activity and thereby limits the production and neurotoxicity of PrP<sup>Sc</sup> or the impact of a TNF α inflammatory insult. As the pharmacological inhibition of ROCK or PDK1 mitigates prion diseases in mice, the ROCK-PDK1 duo emerges as a way to combat prion diseases.

## 192. Prion misfolding activity in ultracentrifugated supernatants of ScN2a cell extracts

Margarita Villavedra and David Cullis-Hill

Sylvan Pharmaceutical Pty. Ltd., Sydney, Australia

CONTACT David Cullis-Hill david@cullis-hill.com

## ABSTRACT

Heparan sulphate proteoglycans (HSPG) are involved in many biological processes. There is evidence that suggests they are involved in the conformational conversion of PrP<sup>C</sup> to the proteinase resistant PrP<sup>Sc</sup>. PrP<sup>Sc</sup> accumulates, together with HSPG, in the cerebral prion amyloid plaques which are a characteristic feature in the pathology of the transmissible spongiform encephalopathies (TSE). Further, depletion of HSPG in N2a cells limited the formation of PrP<sup>Sc</sup>. Also, sulphated glycans like pentosan polysulfate (PPS) showed capacity to protect PrP<sup>C</sup> from its conversion to PrP<sup>Sc</sup> in cell culture. Prion proteins have two heparan binding sites and it has been suggested that the sulphated glycans compete with the HS that induce the misfolding of PrP<sup>C</sup>.

In order to study the role of HSPG in the prion misfolding process we obtained supernatants of ultra-centrifugated extracts of N2a and ScN2a cells solubilized in PBS, 0.5% Triton, 150mM NaCl, 1mM EDTA, pH 7.2. The misfolding capability of these supernatants were tested by RT-QuIC assay using recombinant 112-231 PrP<sup>C</sup> (TPrP) or 23-231PrP<sup>C</sup> (FLPrP).

Our results show that significant misfolding activity persist in the supernatant after centrifugation at 100,000 g for 1h at 4°C. Since PrP<sup>Sc</sup> polymerises into amyloid rods it is expected to be completely restricted to the pellet under these centrifugation conditions. The HSPGs on the other hand are soluble and will remain in the supernatant. Our preliminary results showed that, particularly using FLPrP, the reactivity of some supernatants in the RT-QuIC assay can be similar to that obtained for the original ScN2a homogenates. Although it is not possible to rule out any of the PrP<sup>Sc</sup> being present in the supernatant, its levels would be expected to be very significantly reduced, which does not correspond to our results obtained in the quake assay. We propose that HSPG may play a role in the misfolding activity of this ultracentrifugated supernatant.

## 193. Validating the effect of peptide aptamers on the interaction between Amyloid beta oligomer and Prion protein in vitro and in vivo

Antonia N. Klein, Sabine Gilch

Department of Ecosystem and Public Health, Calgary Prion Research Unit, Faculty of Veterinary Medicine and Hotchkiss Brain Institute, University of Calgary, Calgary, Canada
<figure><img src="image_118.png" /></figure>

## ABSTRACT

The α-secretase TACE exerts a protective role at the neuronal cell surface by catalysing the cleavage of several substrates, including (i) TNF α receptors (TNFRs), which confers physiological sensitivity to TNF α, and (ii) the cellular prion protein PrP<sup>C</sup>, which limits its conversion into pathogenic prions PrP<sup>Sc</sup>. We have assessed whether deregulation of TACE contributes to neurodegeneration in prion diseases.

Using the 1C11 neuronal cell line, primary cultures of neurons and mice infected with prions, we show TACE deficit at the surface of prion-infected cells. Mechanistically, PrP<sup>Sc</sup> promotes the internalization of TACE into caveolin-1-enriched vesicles, which diverts TACE activity away from TNFRs and PrP<sup>C</sup>. This renders neurons highly sensitive to TNF α-associated inflammation and amplifies the production of PrP<sup>Sc</sup> in prion-infected neurons. Whether the neuropathological effect of PrP<sup>Sc</sup>-induced TACE internalization also depends on the uncoupling of the α-secretase from other substrates is currently investigated.

We provide evidence that TACE internalization in prion-infected neurons originates from a loss-of-PrP<sup>C</sup> control of TACE localization at the cell surface upon PrP<sup>C</sup> conversion into PrP<sup>Sc</sup>. The silencing of PrP<sup>C</sup> in uninfected neurons or the conversion of PrP<sup>C</sup> into PrP<sup>Sc</sup> in prion-infected neurons both promote plasma membrane aggregation of β 1 integrins and a rise of β 1 integrin signalling. This leads to the downstream over-activation of the kinase ROCK that complexes with another kinase, PDK1, and overstimulates PDK1 activity. Overactivated PDK1 then triggers the displacement of TACE from the plasma membrane to caveolin 1-enriched vesicles. The inhibition of ROCK or PDK1 is sufficient to target TACE back to the plasma membrane where TACE recovers its protective activity and thereby limits the production and neurotoxicity of PrP<sup>Sc</sup> or the impact of a TNF α inflammatory insult. As the pharmacological inhibition of ROCK or PDK1 mitigates prion diseases in mice, the ROCK-PDK1 duo emerges as a way to combat prion diseases.

## 192. Prion misfolding activity in ultracentrifugated supernatants of ScN2a cell extracts

Margarita Villavedra and David Cullis-Hill

Sylvan Pharmaceutical Pty. Ltd., Sydney, Australia

CONTACT David Cullis-Hill david@cullis-hill.com

## ABSTRACT

Heparan sulphate proteoglycans (HSPG) are involved in many biological processes. There is evidence that suggests they are involved in the conformational conversion of PrP<sup>C</sup> to the proteinase resistant PrP<sup>Sc</sup>. PrP<sup>Sc</sup> accumulates, together with HSPG, in the cerebral prion amyloid plaques which are a characteristic feature in the pathology of the transmissible spongiform encephalopathies (TSE). Further, depletion of HSPG in N2a cells limited the formation of PrP<sup>Sc</sup>. Also, sulphated glycans like pentosan polysulfate (PPS) showed capacity to protect PrP<sup>C</sup> from its conversion to PrP<sup>Sc</sup> in cell culture. Prion proteins have two heparan binding sites and it has been suggested that the sulphated glycans compete with the HS that induce the misfolding of PrP<sup>C</sup>.

In order to study the role of HSPG in the prion misfolding process we obtained supernatants of ultracentrifugated extracts of N2a and ScN2a cells solubilized in PBS, 0.5% Triton, 150mM NaCl, 1mM EDTA, pH 7.2. The misfolding capability of these supernatants were tested by RT-QuIC assay using recombinant 112-231 PrP<sup>C</sup> (TPrP) or 23-231PrP<sup>C</sup> (FLPrP).

Our results show that significant misfolding activity persist in the supernatant after centrifugation at 100,000 g for 1h at 4°C. Since PrP<sup>Sc</sup> polymerises into amyloid rods it is expected to be completely restricted to the pellet under these centrifugation conditions. The HSPGs on the other hand are soluble and will remain in the supernatant. Our preliminary results showed that, particularly using FLPrP, the reactivity of some supernatants in the RT-QuIC assay can be similar to that obtained for the original ScN2a homogenates. Although it is not possible to rule out any of the PrP<sup>Sc</sup> being present in the supernatant, its levels would be expected to be very significantly reduced, which does not correspond to our results obtained in the quake assay. We propose that HSPG may play a role in the misfolding activity of this ultracentrifugated supernatant.

## 193. Validating the effect of peptide aptamers on the interaction between Amyloid beta oligomer and Prion protein in vitro and in vivo

Antonia N. Klein, Sabine Gilch

Department of Ecosystem and Public Health, Calgary Prion Research Unit, Faculty of Veterinary Medicine and Hotchkiss Brain Institute, University of Calgary, Calgary, Canada
<figure><img src="image_119.png" /></figure>

CONTACT Antonia N. Klein Antonia.Klein@ucalgary.ca

ABSTRACT

**Introduction:** Alzheimer's disease (AD) is a neurodegenerative disease that affects 46.8 million people worldwide (estimated in 2015). Aβ monomers aggregates to toxic Aβ oligomers (AβO) and higher aggregates. Studies suggest that the AβO-PrP<sup>C</sup>-interaction at PrP<sup>23-27</sup> (low-affinity side) and PrP<sup>95-110</sup> (high-affinity side) is required for AβO-induced cell death. Previously, we selected peptide aptamers (PA) binding to PrP<sup>C</sup> to inhibit the conformational conversion of PrP<sup>C</sup> to infectious PrP<sup>Sc</sup> associated with prion diseases. These PAs consist of a 16 amino acid peptide motif integrated into bacterial thioredoxin A (trxA) as a scaffold protein. One PA, termed PA8, binds to PrP<sup>C</sup> at position 100-120. Furthermore, we developed PA8 variants, termed 46K, 46Q, and 47H, with single amino acid replacements to improve the binding affinity. The PAs increases total PrP<sup>C</sup> on the cell surface and enhances α-cleavage of PrP<sup>C</sup> into neuroprotective N1- and C1-fragments. The goal of the study presented here was to validate these PAs for treatment of AD by inhibiting the AβO-PrP<sup>C</sup>-interaction.

**Methods:** Synthetic Aβ(1-42) was used to generate AβO, which were characterized by density gradient centrifugation. AβO-PrP<sup>C</sup> interaction was analysed by incubating wtN2A cells with FITC-AβO with or without PA, PrP<sup>C</sup> immunostaining and subsequent analysis of FITC-Aβ and PrP<sup>C</sup> colocalization. Toxicity induced by the AβO-PrP<sup>C</sup>-interaction was analysed by MTT assays and Western blot analysis of active Caspase 3. In the ongoing *in vivo* study, 7 weeks old 5xFAD mice are treated with PA for 12 weeks intraventricularly using osmotic Alzet® pumps. Behavioural tests like novel object recognition test and Morris water maze will be performed within the last treatment week.

**Results:** The here used PAs inhibited AβO-PrP<sup>C</sup>-interaction. Treatment of wtN2A cells with AβO led to a reduced cell viability of 34 ± 7%. Concomitant treatment of wtN2A cells with PA8 and 46K for 24 h resulted in a significant increase of cell viability to 44 ± 8% and 48 ± 16%, respectively. Treating SH-SY5Y cells (not detectable PrP<sup>C</sup> levels) with PAs and AβO, followed by MTT assay confirmed that the enhanced cell viability is PrP<sup>C</sup> dependent. Western blot analysis of active Caspase 3 proved reduced Caspase 3 activation after treatment of wtN2A cells with PAs. Whether these effects ultimately result in memory improvement will be evaluated through the *in vivo* studies.

**Conclusion:** In conclusion, the here used PA were able to inhibit the AβO-induced toxicity *in vitro*. These findings indicate that PAs are interesting candidates for the treatment of both prion diseases and AD.

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

Marie Kostelanskaª, Zdenka Backovska Hanusovaª,
Jakub Soukupª, Radoslav Matejᵇ and Karel Holadaª

ªInstitute of Immunology and Microbiology, First Faculty of Medicine, Charles University and General University Hospital in Prague, Czech Republic; ᵇDepartment of Pathology and Molecular Medicine, Thomayer Teaching Hospital, Prague, Czech Republic

CONTACT Jakub Soukup marie.kostelanska@lf1.cuni.cz

ABSTRACT

**Introduction:** Prions are highly resistant to conventional sterilization procedures and currently recommended inactivation methods include treatment with highly corrosive chemicals like 2% NaClO or 1M NaOH. Such harsh treatment is incompatible with a number of delicate medical tools. We have previously demonstrated that nontoxic disulfonated hydroxyalumminum phthalocyanine (AlPcOH(SO<sub>3</sub>)<sub>2</sub>) can be used to initiate photodynamic inactivation (PDI) of prions at ambient pressure and temperature [1]. The aim of this study was to demonstrate the effectiveness of PDI of prions by bioassay in laboratory mouse.

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

**Results:** Mice inoculated with PDI-treated RML BH (at a 10⁻² dilution) survived significantly longer (204 ± 23 days) than mice inoculated with the homogenate containing an
<figure><img src="image_119.png" /></figure>

CONTACT Antonia N. Klein Antonia.Klein@ucalgary.ca

ABSTRACT

**Introduction:** Alzheimer's disease (AD) is a neurodegenerative disease that affects 46.8 million people worldwide (estimated in 2015). Aβ monomers aggregates to toxic Aβ oligomers (AβO) and higher aggregates. Studies suggest that the AβO-PrP<sup>C</sup>-interaction at PrP<sup>23-27</sup> (low-affinity side) and PrP<sup>95-110</sup> (high-affinity side) is required for AβO-induced cell death. Previously, we selected peptide aptamers (PA) binding to PrP<sup>C</sup> to inhibit the conformational conversion of PrP<sup>C</sup> to infectious PrP<sup>Sc</sup> associated with prion diseases. These PAs consist of a 16 amino acid peptide motif integrated into bacterial thioredoxin A (trxA) as a scaffold protein. One PA, termed PA8, binds to PrP<sup>C</sup> at position 100-120. Furthermore, we developed PA8 variants, termed 46K, 46Q, and 47H, with single amino acid replacements to improve the binding affinity. The PAs increases total PrP<sup>C</sup> on the cell surface and enhances α-cleavage of PrP<sup>C</sup> into neuroprotective N1- and C1-fragments. The goal of the study presented here was to validate these PAs for treatment of AD by inhibiting the AβO-PrP<sup>C</sup>-interaction.

**Methods:** Synthetic Aβ(1-42) was used to generate AβO, which were characterized by density gradient centrifugation. AβO-PrP<sup>C</sup> interaction was analysed by incubating wtN2A cells with FITC-AβO with or without PA, PrP<sup>C</sup> immunostaining and subsequent analysis of FITC-Aβ and PrP<sup>C</sup> colocalization. Toxicity induced by the AβO-PrP<sup>C</sup>-interaction was analysed by MTT assays and Western blot analysis of active Caspase 3. In the ongoing *in vivo* study, 7 weeks old 5xFAD mice are treated with PA for 12 weeks intraventricularly using osmotic Alzet® pumps. Behavioural tests like novel object recognition test and Morris water maze will be performed within the last treatment week.

**Results:** The here used PAs inhibited AβO-PrP<sup>C</sup>-interaction. Treatment of wtN2A cells with AβO led to a reduced cell viability of 34 ± 7%. Concomitant treatment of wtN2A cells with PA8 and 46K for 24 h resulted in a significant increase of cell viability to 44 ± 8% and 48 ± 16%, respectively. Treating SH-SY5Y cells (not detectable PrP<sup>C</sup> levels) with PAs and AβO, followed by MTT assay confirmed that the enhanced cell viability is PrP<sup>C</sup> dependent. Western blot analysis of active Caspase 3 proved reduced Caspase 3 activation after treatment of wtN2A cells with PAs. Whether these effects ultimately result in memory improvement will be evaluated through the *in vivo* studies.

**Conclusion:** In conclusion, the here used PA were able to inhibit the AβO-induced toxicity *in vitro*. These findings indicate that PAs are interesting candidates for the treatment of both prion diseases and AD.

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

Marie Kostelanskaª, Zdenka Backovska Hanusovaª,
Jakub Soukupª, Radoslav Matejᵇ and Karel Holadaª

ªInstitute of Immunology and Microbiology, First Faculty of Medicine, Charles University and General University Hospital in Prague, Czech Republic; ᵇDepartment of Pathology and Molecular Medicine, Thomayer Teaching Hospital, Prague, Czech Republic

CONTACT Jakub Soukup marie.kostelanska@lf1.cuni.cz

ABSTRACT

**Introduction:** Prions are highly resistant to conventional sterilization procedures and currently recommended inactivation methods include treatment with highly corrosive chemicals like 2% NaClO or 1M NaOH. Such harsh treatment is incompatible with a number of delicate medical tools. We have previously demonstrated that nontoxic disulfonated hydroxyaluminum phthalocyanine (AlPcOH(SO<sub>3</sub>)<sub>2</sub>) can be used to initiate photodynamic inactivation (PDI) of prions at ambient pressure and temperature [1]. The aim of this study was to demonstrate the effectiveness of PDI of prions by bioassay in laboratory mouse.

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

**Results:** Mice inoculated with PDI-treated RML BH (at a 10⁻² dilution) survived significantly longer (204 ± 23 days) than mice inoculated with the homogenate containing an
<figure><img src="image_120.png" /></figure>

identical amount of AlPcOH(SO₃)₂, that was not exposed to light (165 ± 11 days). Their survival was also significantly longer than that of mice inoculated with straight 10⁻², 10⁻³, and 10⁻⁴ dilutions of the RML BH, who survived 169 ± 11, 165 ± 12, and 176 ± 15 days, respectively. The transmission rate was 100% in all groups of mice including serial dilution (10⁻² to 10⁻⁶). A comparison of survival time based on the regression line suggested a decrease in the prion infectivity titer of the RML BH of more than four orders of magnitude after PDI treatment.

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

**KEYWORDS:** Prion; phthalocyanine; photodynamic inactivation; decontamination; mouse bioassay
The project was supported by AZV NV18-04-00179.

## Reference

[1] Janouskova et al., J Gen Virol. 2012; 93(Pt11): 2512-2517.

# 195. When anchorage determines fate: How the GPI-anchor signal sequence impacts on prion protein biology and prion disease in mice

Berta Puig<sup>a,b</sup>, Hermann C. Altmeppen<sup>a</sup>, Luise Linsenmeier<sup>a</sup>, Karima Chakroun<sup>a</sup>, Ulrike K. Piontek<sup>c</sup>, Jörg Tatzelt<sup>c,d</sup>, Clive Bate<sup>e</sup>, Tim Magnus<sup>b</sup> and Markus Glatzel<sup>a</sup>

<sup>a</sup>Institute of Neuropathology; <sup>b</sup>Department of Neurology, Experimental Research in Stroke and Inflammation (ERSI), University Medical Center Hamburg-Eppendorf (UKE), Hamburg, Germany; <sup>c</sup>Adolf Butenandt Institute, Ludwig Maximilians University, Munich, Germany; <sup>d</sup>Institute of Biochemistry and Pathobiochemistry, Biochemistry of Neurodegenerative Diseases, Ruhr University, Bochum, Germany; <sup>e</sup>Department of Pathology and Pathogen Biology, Royal Veterinary College, Hawkshead Lane, North Mymms, Herts, UK

**CONTACT** Hermann C. Altmeppen h.altmeppen@uke.de

## ABSTRACT

Correct anchoring and localization of the prion protein (PrP) at the neuronal surface are essential for its processing, functions and roles in disease. Several studies have revealed a compensatory network regulating PrP membrane homeostasis, including proteolytic or extracellular vesicle release, and internalization followed by retrograde transport and degradation. In this study however, we asked if the GPI-anchor signal sequence (GPI-SS) -though not

part of the mature protein- affects composition, trafficking, membrane localization and, hence, biology of PrP in the first place, and whether alterations in that regard would impact on prion disease in particular. We generated different lines of transgenic mice expressing a mutant PrP (PrP<sup>C</sup>GPIThy-1) containing the GPI-SS of the neuronal protein Thy-1, a resident of dense cores of lipid rafts. This modification indeed resulted in a different GPI-anchor composition (now lacking the rare mammalian sialic acid typical for PrP), altered sorting in polarized cells (such as primary neurons), and reduced proteolytic shedding by the metalloprotease ADAM10. When inoculated with either RML or 22L prions, survival of mice was significantly prolonged for all transgenic lines, despite similar or even higher expression levels of generally signalling- and misfolding-competent PrP<sup>C</sup>GPIThy-1 compared to wild-type controls. Fittingly, MAP kinase signalling as well as glial activation and spongiform lesions were reduced in brains of our transgenic mice. Using PrP as a model, this is the first *in vivo* study demonstrating that the GPI-SS determines the later remodelling and composition of a given protein's GPI-anchor and sorting, with likely consequences for its biology. Besides drastic effects on different pathomechanistic aspects underlying the course of prion diseases, our findings may also have wider implications for the field of GPI-anchor biology.

**KEYWORDS:** ADAM10; glycosylphosphatidylinositol (GPI)-anchor; lipid rafts; prion protein; shedding; signalling; sorting

## Reference

[1] Puig, Altmeppen et al., PLoS Path. 2019.

# 196. Reverting prion disease in neuronal progenitor cells as a cell replacement therapy

Arielle Hay, Lindsay Parrie, Glenn C. Telling, Mark Zabel and Julie A. Moreno

Prion Research Center, Department of Microbiology, Immunology & Pathology, Colorado State University, Fort Collins, CO, USA

**CONTACT** Arielle Hay Arielle.Hay@colostate.edu

## ABSTRACT

We propose the use of gene edited stem cells as therapy for neurodegenerative diseases. Olfactory neuronal progenitors (ONPs) can differentiate into neurons in adulthood. We hypothesize they will
<figure><img src="image_120.png" /></figure>

identical amount of AlPcOH(SO₃)₂, that was not exposed to light (165 ± 11 days). Their survival was also significantly longer than that of mice inoculated with straight 10⁻², 10⁻³, and 10⁻⁴ dilutions of the RML BH, who survived 169 ± 11, 165 ± 12, and 176 ± 15 days, respectively. The transmission rate was 100% in all groups of mice including serial dilution (10⁻² to 10⁻⁶). A comparison of survival time based on the regression line suggested a decrease in the prion infectivity titer of the RML BH of more than four orders of magnitude after PDI treatment.

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

**KEYWORDS:** Prion; phthalocyanine; photodynamic inactivation; decontamination; mouse bioassay
The project was supported by AZV NV18-04-00179.

## Reference

[1] Janouskova et al., J Gen Virol. 2012; 93(Pt11): 2512-2517.

# 195. When anchorage determines fate: How the GPI-anchor signal sequence impacts on prion protein biology and prion disease in mice

Berta Puig<sup>a,b</sup>, Hermann C. Altmeppen<sup>a</sup>, Luise Linsenmeier<sup>a</sup>, Karima Chakroun<sup>a</sup>, Ulrike K. Piontek<sup>c</sup>, Jörg Tatzelt<sup>c,d</sup>, Clive Bate<sup>e</sup>, Tim Magnus<sup>b</sup> and Markus Glatzel<sup>a</sup>

<sup>a</sup>Institute of Neuropathology; <sup>b</sup>Department of Neurology, Experimental Research in Stroke and Inflammation (ERSI), University Medical Center Hamburg-Eppendorf (UKE), Hamburg, Germany; <sup>c</sup>Adolf Butenandt Institute, Ludwig Maximilians University, Munich, Germany; <sup>d</sup>Institute of Biochemistry and Pathobiochemistry, Biochemistry of Neurodegenerative Diseases, Ruhr University, Bochum, Germany; <sup>e</sup>Department of Pathology and Pathogen Biology, Royal Veterinary College, Hawkshead Lane, North Mymms, Herts, UK

**CONTACT** Hermann C. Altmeppen h.altmeppen@uke.de

## ABSTRACT

Correct anchoring and localization of the prion protein (PrP) at the neuronal surface are essential for its processing, functions and roles in disease. Several studies have revealed a compensatory network regulating PrP membrane homeostasis, including proteolytic or extracellular vesicle release, and internalization followed by retrograde transport and degradation. In this study however, we asked if the GPI-anchor signal sequence (GPI-SS) -though not

part of the mature protein- affects composition, trafficking, membrane localization and, hence, biology of PrP in the first place, and whether alterations in that regard would impact on prion disease in particular. We generated different lines of transgenic mice expressing a mutant PrP (PrP<sup>C</sup>GPIThy-1) containing the GPI-SS of the neuronal protein Thy-1, a resident of dense cores of lipid rafts. This modification indeed resulted in a different GPI-anchor composition (now lacking the rare mammalian sialic acid typical for PrP), altered sorting in polarized cells (such as primary neurons), and reduced proteolytic shedding by the metalloprotease ADAM10. When inoculated with either RML or 22L prions, survival of mice was significantly prolonged for all transgenic lines, despite similar or even higher expression levels of generally signalling- and misfolding-competent PrP<sup>C</sup>GPIThy-1 compared to wild-type controls. Fittingly, MAP kinase signalling as well as glial activation and spongiform lesions were reduced in brains of our transgenic mice. Using PrP as a model, this is the first *in vivo* study demonstrating that the GPI-SS determines the later remodelling and composition of a given protein's GPI-anchor and sorting, with likely consequences for its biology. Besides drastic effects on different pathomechanistic aspects underlying the course of prion diseases, our findings may also have wider implications for the field of GPI-anchor biology.

**KEYWORDS:** ADAM10; glycosylphosphatidylinositol (GPI)-anchor; lipid rafts; prion protein; shedding; signalling; sorting

## Reference

[1] Puig, Altmeppen et al., PLoS Path. 2019.

# 196. Reverting prion disease in neuronal progenitor cells as a cell replacement therapy

Arielle Hay, Lindsay Parrie, Glenn C. Telling, Mark Zabel and Julie A. Moreno

Prion Research Center, Department of Microbiology, Immunology & Pathology, Colorado State University, Fort Collins, CO, USA

**CONTACT** Arielle Hay Arielle.Hay@colostate.edu

## ABSTRACT

We propose the use of gene edited stem cells as therapy for neurodegenerative diseases. Olfactory neuronal progenitors (ONPs) can differentiate into neurons in adulthood. We hypothesize they will
regenerate neurons that have been lost due to aggre-
gation of disease- associated proteins. Mesenchymal
stem cells (MSCs) can be derived from adipocytes of
adult mice and further differentiated to neural stem
cells (NSCs), which we plan to use as an alternative
to ONPs. These therapies will be modelled by prion
infected mice, which display the typical features of
neurodegenerative disease, including clinical signs
and loss of neurons. Prion diseases are the result of
misfolding of the prion protein (PrP), which is highly
expressed in neurons. PrP knockout mice are resis-
tant to prion diseases. Two populations of ONPs will
be made using CRISPR/Cas9 gene editing to delete
the *prnp* gene in N2a cells and WT ONPs. These cells
will be subjected to prion cell assays to determine if
they can be infected. The second population of ONPs
will be from mice with a mutation associated with
the human prion disease Gerstmann-Sträussler-
Scheinker syndrome (GSS), which is modelled in
mice by a proline to leucine switch at codon 101 of
moPrP. We plan to use CRISPR/Cas9 to revert this
mutation. Alternatively, we plan to edit the *prnp* gene
in M/NSCs so they express a secretable, dominant-
negative PrP. Single cell sorting will be used to pro-
duce homogenous populations of edited cells, which
will be sequenced to identify relevant mutations. We
will engraft gene edited ONPs using stereotactic
injection and M/NSCs using nasal instillation into
the brains of prion infected WT and GSS transgenic
mice [1]. We hypothesize that these cells will resist
prion infection. ONPs will migrate throughout the
brain to restore damaged neurons and M/NSCs will
 populate the olfactory bulb and secrete dominant
negative PrP and anti-inflammatory cytokines
throughout the brain.

197. Proteolytic shedding of PrP can be
manipulated in a substrate-specific manner by
certain PrP-binding ligands

Luise Linsenmeier<sup>a</sup>, Behnam Mohammadi<sup>a</sup>, Mohsin
Shafiq<sup>a</sup>, Simrika Thapa<sup>b</sup>, Karl Frontzek<sup>c</sup>, Berta Puig<sup>a</sup>,
Chaoyang Li<sup>d</sup>, Michaela Schweizer<sup>e</sup>, Tania Massignan<sup>f</sup>,
Emiliano Biasini<sup>f</sup>, Antonia Klein<sup>b</sup>, Sabine Gilch<sup>b</sup>, Luca
Varani<sup>g</sup>, Hermann Schatzl<sup>b</sup>, Adriano Aguzzi<sup>c</sup>,
Hermann C. Altmeppen<sup>a*</sup> and Markus Glatzel<sup>a*</sup>

Medical Center HH-Eppendorf, Hamburg, Germany; ªDepartment
of Cellular, Computational and Integrative Biology (CIBIO),
University of Trento, Italy; gInstitute for Research in Biomedicine,
Università della Svizzera italiana, Bellinzona, Switzerland

*These authors contributed equally to this work.

CONTACT Luise Linsenmeier l.linsenmeier@uke.de

ABSTRACT

The prion protein (PrP<sup>C</sup>) impacts on development and
maintenance of the nervous system and plays a deleter-
ious role in fatal and progressive prion diseases.
Moreover, in other neurodegenerative proteinopathies
it acts as a receptor for toxic oligomers such as A&beta; in
Alzheimer's disease (AD). PrP<sup>C</sup> is released from the cell
surface into the extracellular space by the metallopro-
tease ADAM10. We have shown that this cleavage sig-
nificantly impacts on prion disease, and soluble PrP
fragments may be key in understanding the multitude
of functions suggested for PrP<sup>C</sup>, such as in myelin
maintenance, neuroprotection, synapse formation or
neuroimmune crosstalk.

We expect the ADAM10-mediated shedding of PrP<sup>C</sup>
to have therapeutic potential against neurodegenerative
proteinopathies. However, a substrate-specific
approach to stimulate this cleavage would likely be
superior to targeting of the protease, given the plethora
of ADAM10 substrates throughout the body and, thus,
the risk of severe side-effects. We found that several
PrP-directed antibodies significantly increase shedding
in different cell culture models and organotypic slice
cultures. Crosslinking of PrP is not a prerequisite since
treatment with single chain antibodies and other PrP-
binders likewise resulted in increased cleavage. We thus
assume that binding of certain ligands to PrP per se
stimulates its shedding. While we also identified some
antibodies that blocked shedding, one particular anti-
body (likely due to its unique binding characteristics)
led to strong surface clustering, fast internalization and
degradation of PrP rather than to proteolytic release
from the membrane.

Of note, PrP-directed antibodies or compounds have
been experimentally suggested or are currently tested as
a treatment option in fatal neurodegenerative proteino-
pathies. It remains to be investigated whether stimu-
lated shedding is (at least partially) underlying the
protective mechanism. In contrast, given the recently
suggested harmful roles of shed PrP in brain cancer and
HIV-associated neuropathogenesis, the shedding-inhi-
biting ligands identified in our study may also hold
therapeutic potential in pathological conditions affect-
ing the brain.

<footnote><sup>a</sup>Institute of Neuropathology, University Medical Center HH-Eppendorf, Hamburg, Germany; <sup>b</sup>Calgary Prion Research Unit, Faculty of Veterinary Medicine, University of Calgary, Canada; <sup>c</sup>Institute of Neuropathology, University Hospital Zurich, Switzerland; <sup>d</sup>Wuhan Institute of Virology, Chinese Academy of Science, Beijing, China; <sup>e</sup>Center for Molecular Neurobiology Hamburg, Morphology and Electron Microscopy Unit, University Medical Center HH-Eppendorf, Hamburg, Germany; <sup>f</sup>Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Italy; <sup>g</sup>Institute for Research in Biomedicine, Università della Svizzera italiana, Bellinzona, Switzerland</footnote>
regenerate neurons that have been lost due to aggre-
gation of disease- associated proteins. Mesenchymal
stem cells (MSCs) can be derived from adipocytes of
adult mice and further differentiated to neural stem
cells (NSCs), which we plan to use as an alternative
to ONPs. These therapies will be modelled by prion
infected mice, which display the typical features of
neurodegenerative disease, including clinical signs
and loss of neurons. Prion diseases are the result of
misfolding of the prion protein (PrP), which is highly
expressed in neurons. PrP knockout mice are resis-
tant to prion diseases. Two populations of ONPs will
be made using CRISPR/Cas9 gene editing to delete
the *prnp* gene in N2a cells and WT ONPs. These cells
will be subjected to prion cell assays to determine if
they can be infected. The second population of ONPs
will be from mice with a mutation associated with
the human prion disease Gerstmann-Sträussler-
Scheinker syndrome (GSS), which is modelled in
mice by a proline to leucine switch at codon 101 of
moPrP. We plan to use CRISPR/Cas9 to revert this
mutation. Alternatively, we plan to edit the *prnp* gene
in M/NSCs so they express a secretable, dominant-
negative PrP. Single cell sorting will be used to pro-
duce homogenous populations of edited cells, which
will be sequenced to identify relevant mutations. We
will engraft gene edited ONPs using stereotactic
injection and M/NSCs using nasal instillation into
the brains of prion infected WT and GSS transgenic
mice [1]. We hypothesize that these cells will resist
prion infection. ONPs will migrate throughout the
brain to restore damaged neurons and M/NSCs will
 populate the olfactory bulb and secrete dominant
negative PrP and anti-inflammatory cytokines
throughout the brain.

197. Proteolytic shedding of PrP can be
manipulated in a substrate-specific manner by
certain PrP-binding ligands

Luise Linsenmeier<sup>a</sup>, Behnam Mohammadi<sup>a</sup>, Mohsin
Shafiq<sup>a</sup>, Simrika Thapa<sup>b</sup>, Karl Frontzek<sup>c</sup>, Berta Puig<sup>a</sup>,
Chaoyang Li<sup>d</sup>, Michaela Schweizer<sup>e</sup>, Tania Massignan<sup>f</sup>,
Emiliano Biasini<sup>f</sup>, Antonia Klein<sup>b</sup>, Sabine Gilch<sup>b</sup>, Luca
Varani<sup>g</sup>, Hermann Schatzl<sup>b</sup>, Adriano Aguzzi<sup>c</sup>,
Hermann C. Altmeppen<sup>a*</sup> and Markus Glatzel<sup>a*</sup>

Medical Center HH-Eppendorf, Hamburg, Germany; †Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Italy; ‡Institute for Research in Biomedicine, Università della Svizzera italiana, Bellinzona, Switzerland

*These authors contributed equally to this work.

CONTACT Luise Linsenmeier l.linsenmeier@uke.de

ABSTRACT

The prion protein (PrP<sup>C</sup>) impacts on development and
maintenance of the nervous system and plays a deleter-
ious role in fatal and progressive prion diseases.
Moreover, in other neurodegenerative proteinopathies
it acts as a receptor for toxic oligomers such as A&beta; in
Alzheimer's disease (AD). PrP<sup>C</sup> is released from the cell
surface into the extracellular space by the metallopro-
tease ADAM10. We have shown that this cleavage sig-
nificantly impacts on prion disease, and soluble PrP
fragments may be key in understanding the multitude
of functions suggested for PrP<sup>C</sup>, such as in myelin
maintenance, neuroprotection, synapse formation or
neuroimmune crosstalk.

We expect the ADAM10-mediated shedding of PrP<sup>C</sup>
to have therapeutic potential against neurodegenerative
proteinopathies. However, a substrate-specific
approach to stimulate this cleavage would likely be
superior to targeting of the protease, given the plethora
of ADAM10 substrates throughout the body and, thus,
the risk of severe side-effects. We found that several
PrP-directed antibodies significantly increase shedding
in different cell culture models and organotypic slice
cultures. Crosslinking of PrP is not a prerequisite since
treatment with single chain antibodies and other PrP-
binders likewise resulted in increased cleavage. We thus
assume that binding of certain ligands to PrP per se
stimulates its shedding. While we also identified some
antibodies that blocked shedding, one particular anti-
body (likely due to its unique binding characteristics)
led to strong surface clustering, fast internalization and
degradation of PrP rather than to proteolytic release
from the membrane.

Of note, PrP-directed antibodies or compounds have
been experimentally suggested or are currently tested as
a treatment option in fatal neurodegenerative proteino-
pathies. It remains to be investigated whether stimu-
lated shedding is (at least partially) underlying the
protective mechanism. In contrast, given the recently
suggested harmful roles of shed PrP in brain cancer and
HIV-associated neuropathogenesis, the shedding-inhi-
biting ligands identified in our study may also hold
therapeutic potential in pathological conditions affect-
ing the brain.

<footnote><sup>a</sup>Institute of Neuropathology, University Medical Center HH-Eppendorf, Hamburg, Germany; <sup>b</sup>Calgary Prion Research Unit, Faculty of Veterinary Medicine, University of Calgary, Canada; <sup>c</sup>Institute of Neuropathology, University Hospital Zurich, Switzerland; <sup>d</sup>Wuhan Institute of Virology, Chinese Academy of Science, Beijing, China; <sup>e</sup>Center for Molecular Neurobiology Hamburg, Morphology and Electron Microscopy Unit, University Medical Center HH-Eppendorf, Hamburg, Germany; <sup>f</sup>Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Italy; <sup>g</sup>Institute for Research in Biomedicine, Università della Svizzera italiana, Bellinzona, Switzerland</footnote>
<figure><img src="image_121.png" /></figure>

# 198. Predicting Blood-Brain Partitioning and Skin Permeability using 3D-RISM-KH Theory Based Solvation Energy Descriptors

Andriy Kovalenko

Department of Mechanical Engineering, University of Alberta, 10-203 Donadeo Innovation Centre for Engineering, Edmonton, Alberta, Canada; Nanotechnology Research Centre, Edmonton, Alberta, Canada

## ABSTRACT

Compartmentalization of drug molecules between plasma and brain is important for desired activity. The difficulty in obtaining the blood-brain permeability of drug (like) substances experimentally resulted in the development of several theoretical quantitative structure activity relationships (QSAR) towards predicting capability of a given substrate to pass across tight junction, known as the blood-brain barrier, both qualitatively and quantitatively. We have developed a novel method based on the three-dimensional reference interaction site model with the Kovalenko-Hirata closure relation (3D-RISM-KH) molecular solvation theory for predicting blood-brain barrier permeability coefficients of molecules in diverse structures with a minimum set of descriptors derived from solvation energetics [1]. Our findings reveal the importance of solvation free energy based descriptors in quantitatively modelling blood-brain barrier permeability. Further, we use the state-of-the-art molecular solvation theory to predict skin permeability of a large set of compounds with experimental logKP [2]. We obtained encouraging results pointing to applicability of the model using statistical physics based 3D-RISM-KH theory for solvation free energy calculations as a primary descriptor with relative mean square error of 0.77 units.

## References

[1] Roy D, Hinge VK, Kovalenko A. Predicting blood-brain partitioning of small molecules using a novel minimalistic descriptor based approach via the 3D-RISM-KH molecular solvation theory. *ACS Omega*. 2019 (accepted).

[2] Hinge VK, Roy D, Kovalenko A. Predicting skin permeability using the 3D-RISM-KH theory based solvation energy descriptors for a diverse class of compounds. *J Pharm Sci*, 2019 (submitted).

# 199. Chronic wasting disease transmission studies to non-human primates and transgenic mice

Brent Race, Katie Williams, Christina D. Orrù, Andrew G. Hughson, Lori Lubke and Bruce Chesebro

National Institute of Allergy and Infectious Diseases, Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, Hamilton, MT, USA

CONTACT Brent Race raceb@niaid.nih.gov

## ABSTRACT

**Introduction:** Chronic wasting disease (CWD) is the only prion disease affecting free-ranging animals, reported in North America, South Korea and Norway. Unlike in most other prion disease CWD agents are shed in blood, urine and feces which most likely contribute to the horizontal transmission between cervid species. The developments of amplification-based seeding assays have been instrumental in the detection of low levels of prions in clinical samples. Using real-time quaking-induced conversion (RT-QUIC), we established an ultrasensitive detection method for PrPCWD in the urine from CWD-infected sequentially sampled transgenic mice overexpressing elk prion protein (TgElk mice). In addtion, RT-QUIC was performed in the kidney and brain of theses mice model to trace abnormal prion.

**Materials and Methods:** 44 brain and kidney, urine samples from sequentially collected from CWD-infected TgElk mice (TgElk CWD) were stored at -80°C. In brain and kidney, 10% (w/v) homogenate was prepared in 0.9% sterilized saline. In urine 100uL of each sample was mixed with 10uL 2.8% sodium phosphotungustic acid (NaPTA) and incubated for 1hr at 37°C with shaking at 1,350 rpm. Samples were centrifuged for 30min at 16,100g. The pellet was resuspended in 10uL of 0.1% SDS/PBS for 30min at 55°C. RT-QUIC reactions were set up in 96-well clear bottom optic plates and consisted of 98uL RT-QUIC buffer [final concentrations of 1XPBS, 1mM EDTA, 10uM Thioflavin, 300mM NaCl buffer and 0.1mg/ml recombinant Syrian hamster recombinant protein (23-231) and 2uL of sample. The RT-QUIC assay was performed on a FLUOstar Omega fluorescence plate reader that was preheated to 42°C for 60hr with 90sec shaking at 700rpm followed by 1min incubation.

**Results:** Five randomly selected mice were sequentially culled on every 15 days from 30dpi to 120dpi during CWD infected TgElk mice reached terminal stage. Rough hair coats among clinical signs were showed from 90 dpi. PrPCWD in the brain in TgElk CWD was detectable persistently from early stages (30dpi), and in the kidney PrPCWD was also detectable in clinical and terminal stages (90 dpi and 120dpi). PrPCWD in the urine in TgElk CWD reached the highest levels at 120dpi. NaPTA/RT-QUIC was applied to measure PrPCWD in urine samples collected on every 15 days from 30dpi to 120dpi when CWD infected TgElk mice
<figure><img src="image_121.png" /></figure>

# 198. Predicting Blood-Brain Partitioning and Skin Permeability using 3D-RISM-KH Theory Based Solvation Energy Descriptors

Andriy Kovalenko

Department of Mechanical Engineering, University of Alberta, 10-203 Donadeo Innovation Centre for Engineering, Edmonton, Alberta, Canada; Nanotechnology Research Centre, Edmonton, Alberta, Canada

## ABSTRACT

Compartmentalization of drug molecules between plasma and brain is important for desired activity. The difficulty in obtaining the blood-brain permeability of drug (like) substances experimentally resulted in the development of several theoretical quantitative structure activity relationships (QSAR) towards predicting capability of a given substrate to pass across tight junction, known as the blood-brain barrier, both qualitatively and quantitatively. We have developed a novel method based on the three-dimensional reference interaction site model with the Kovalenko-Hirata closure relation (3D-RISM-KH) molecular solvation theory for predicting blood-brain barrier permeability coefficients of molecules in diverse structures with a minimum set of descriptors derived from solvation energetics [1]. Our findings reveal the importance of solvation free energy based descriptors in quantitatively modelling blood-brain barrier permeability. Further, we use the state-of-the-art molecular solvation theory to predict skin permeability of a large set of compounds with experimental logKP [2]. We obtained encouraging results pointing to applicability of the model using statistical physics based 3D-RISM-KH theory for solvation free energy calculations as a primary descriptor with relative mean square error of 0.77 units.

## References

[1] Roy D, Hinge VK, Kovalenko A. Predicting blood-brain partitioning of small molecules using a novel minimalistic descriptor based approach via the 3D-RISM-KH molecular solvation theory. *ACS Omega*. 2019 (accepted).

[2] Hinge VK, Roy D, Kovalenko A. Predicting skin permeability using the 3D-RISM-KH theory based solvation energy descriptors for a diverse class of compounds. *J Pharm Sci*, 2019 (submitted).

# 199. Chronic wasting disease transmission studies to non-human primates and transgenic mice

Brent Race, Katie Williams, Christina D. Orrù, Andrew G. Hughson, Lori Lubke and Bruce Chesebro

National Institute of Allergy and Infectious Diseases, Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, Hamilton, MT, USA

CONTACT Brent Race raceb@niaid.nih.gov

## ABSTRACT

**Introduction:** Chronic wasting disease (CWD) is the only prion disease affecting free-ranging animals, reported in North America, South Korea and Norway. Unlike in most other prion disease CWD agents are shed in blood, urine and feces which most likely contribute to the horizontal transmission between cervid species. The developments of amplification-based seeding assays have been instrumental in the detection of low levels of prions in clinical samples. Using real-time quaking-induced conversion (RT-QUIC), we established an ultrasensitive detection method for PrPCWD in the urine from CWD-infected sequentially sampled transgenic mice overexpressing elk prion protein (TgElk mice). In addtion, RT-QUIC was performed in the kidney and brain of theses mice model to trace abnormal prion.

**Materials and Methods:** 44 brain and kidney, urine samples from sequentially collected from CWD-infected TgElk mice (TgElk CWD) were stored at -80°C. In brain and kidney, 10% (w/v) homogenate was prepared in 0.9% sterilized saline. In urine 100uL of each sample was mixed with 10uL 2.8% sodium phosphotungustic acid (NaPTA) and incubated for 1hr at 37°C with shaking at 1,350 rpm. Samples were centrifuged for 30min at 16,100g. The pellet was resuspended in 10uL of 0.1% SDS/PBS for 30min at 55°C. RT-QUIC reactions were set up in 96-well clear bottom optic plates and consisted of 98uL RT-QUIC buffer [final concentrations of 1XPBS, 1mM EDTA, 10uM Thioflavin, 300mM NaCl buffer and 0.1mg/ml recombinant Syrian hamster recombinant protein (23-231) and 2uL of sample. The RT-QUIC assay was performed on a FLUOstar Omega fluorescence plate reader that was preheated to 42°C for 60hr with 90sec shaking at 700rpm followed by 1min incubation.

**Results:** Five randomly selected mice were sequentially culled on every 15 days from 30dpi to 120dpi during CWD infected TgElk mice reached terminal stage. Rough hair coats among clinical signs were showed from 90 dpi. PrPCWD in the brain in TgElk CWD was detectable persistently from early stages (30dpi), and in the kidney PrPCWD was also detectable in clinical and terminal stages (90 dpi and 120dpi). PrPCWD in the urine in TgElk CWD reached the highest levels at 120dpi. NaPTA/RT-QUIC was applied to measure PrPCWD in urine samples collected on every 15 days from 30dpi to 120dpi when CWD infected TgElk mice
<figure><img src="image_122.png" /></figure>

reached terminal stage. PrPCWD in the urine in TgElk CWD reached the highest levels at 90dpi. PrPCWD was also detectable in late and terminal stages (120dpi).

**Conclusions:** We demonstrate that CWD prions can be detected by RT-QUIC or NaPTA/RT-QUIC in the brain, kidney and urine of TgElk mice at the early and terminal stages of disease. Based on these data, we suggest that PrPCWD is excreted into only urine until 90 dpi and then slowly accumulated in kidney. Our results can be used in designing future study of CWD pathogenesis in TgElk mice.

**KEYWORDS:** Cynomolgus macaques; squirrel monkeys; non-human primate; transgenic mice; CWD; RT-QuIC; prion; cross-species transmission; barrier; chronic wasting disease

## References

[1] Henderson DM et al Rapid antemortem detection of CWD prions in deer saliva PLOS one 2013, 397, 626-630.

[2] Nagooka K, Yoshika M, Shimozaki N. et al. Sensitive detection of scrapie prion protein in soil. Biochem Biophys Res Commun. 2010 e74377 1-12.

## 200. DNA methylation array based machine learning classification of human prion disease

Fernando Guntoro, Luke Dabin, John Collinge, Emmanuelle Vire, and Simon Mead

MRC Prion Unit at UCL, UCL Institute of Prion Diseases, London, UK

CONTACT Fernando Guntoro f.guntoro@ucl.ac.uk

## ABSTRACT

**Background:** Variant Creutzfeldt-Jakob disease (vCJD) was caused by the widespread dietary exposure of the United Kingdom (UK) and other populations to bovine spongiform encephalopathy (BSE) prions. Unlike other forms of CJD, abnormal prion proteins (PrP) in vCJD are readily detectable by conventional histopathology outside the central nervous system, particularly in lymphoreticular system (LRS) tissues such as tonsillar and appendix tissues. To date, six prevalence studies have been conducted; the fifth (Appendix II) and sixth (Appendix III) studies reported abnormal PrP immunoreactivity in 16 out of 32,441 appendix samples and 2 out of 14,692 samples, respectively, indicating a surprisingly high prevalence abnormal PrP. Given the 178 vCJD cases hitherto reported in the UK, there appears to be a discordance between the population prevalence data and the number of observed clinical cases, which challenges the assumption of a perfect association

between PrP deposition in LRS tissues and vCJD disease. To provide insight into this discordance, we aim to develop a classifier based on epigenomic profiles that distinguishes prion infection from healthy controls.

**Materials and Methods:** A unique archive of fixed and frozen LRS tissues including tonsil and appendix from vCJD patients, blood and brain samples from a range of human prion disease patients. Samples have been age/ sex-matched to controls not diagnosed with neurodegenerative disease. For DNA methylation profiling, extracted genomic DNA has been subjected to bisulfite conversion and, subsequently, hybridized to the Illumina 450K or 850K EPIC arrays. Training of a deep learning neural network model was performed using these samples.

**Results:** Initial results explored the diagnostic potential of an Illumina 450K dataset using deep learning neural network classifier. This DNA methylation study was conducted using whole bloods from 106 healthy controls and 114 sCJD patients (age/ sex-matched). Using data from the 1000 most significantly altered methylation loci, this data was randomized and partitioned into training and test set of 1:1 case-control ratio. After 10-fold validation, the model had an accuracy of 85.64% (± 1.14%) with an upper limit of 90.00% accuracy. The trained neural network model demonstrated a better performance compared to a basic Random Forest classifier with an AUC of 0.965 and 0.841, respectively.

**Conclusions:** As a proof of concept, we have developed an accurate classification tool using relatively small numbers of differentially methylated loci in blood. We are extending these results to a wider panel of neurodegenerative dementias and LRS tissues.

**KEYWORDS:** Epigenetics; EWAS; DNA methylation; Variant Creutzfeldt-Jakob; Machine learning; Appendix; Tonsil; Lymphoreticular tissue

## 201. TDP-43 induces SOD1 aggregation and toxicity via tryptophan interactions that present a novel drug target

Michèle G. DuValª, Edward Pokrishevskyᵇ, Vijaya K. Hingeᶜ,>d, Jenna Bratvoldª, Richard Kanyoª,ᵉ, Natalie Snyderª, Nikolay Blinovᶜ,>d, Andriy Kovalenkoᶜ,>d, Neil R. Cashmanᵇ and W. Ted Allisonª,ᵉ,f

<footnote>ªDepartment of Biological Sciences, University of Alberta, Edmonton, AB, Canada; ᵇDjavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, BC, Canada; ᶜNational Institute for Nanotechnology, Edmonton, AB, Canada; ᵈDepartment of Mechanical Engineering, University of Alberta, Edmonton, AB, Canada; ᵉCentre for Prions and Protein Folding Diseases, University</footnote>
reached terminal stage. PrPCWD in the urine in TgElk CWD reached the highest levels at 90dpi. PrPCWD was also detectable in late and terminal stages (120dpi).

**Conclusions:** We demonstrate that CWD prions can be detected by RT-QUIC or NaPTA/RT-QUIC in the brain, kidney and urine of TgElk mice at the early and terminal stages of disease. Based on these data, we suggest that PrPCWD is excreted into only urine until 90 dpi and then slowly accumulated in kidney. Our results can be used in designing future study of CWD pathogenesis in TgElk mice.

**KEYWORDS:** Cynomolgus macaques; squirrel monkeys; non-human primate; transgenic mice; CWD; RT-QuIC; prion; cross-species transmission; barrier; chronic wasting disease

## References

[1] Henderson DM et al Rapid antemortem detection of CWD prions in deer saliva PLOS one 2013, 397, 626-630.

[2] Nagooka K, Yoshika M, Shimozaki N. et al. Sensitive detection of scrapie prion protein in soil. Biochem Biophys Res Commun. 2010 e74377 1-12.

## 200. DNA methylation array based machine learning classification of human prion disease

Fernando Guntoro, Luke Dabin, John Collinge, Emmanuelle Vire, and Simon Mead

MRC Prion Unit at UCL, UCL Institute of Prion Diseases, London, UK

CONTACT Fernando Guntoro f.guntoro@ucl.ac.uk

## ABSTRACT

**Background:** Variant Creutzfeldt-Jakob disease (vCJD) was caused by the widespread dietary exposure of the United Kingdom (UK) and other populations to bovine spongiform encephalopathy (BSE) prions. Unlike other forms of CJD, abnormal prion proteins (PrP) in vCJD are readily detectable by conventional histopathology outside the central nervous system, particularly in lymphoreticular system (LRS) tissues such as tonsillar and appendix tissues. To date, six prevalence studies have been conducted; the fifth (Appendix II) and sixth (Appendix III) studies reported abnormal PrP immunoreactivity in 16 out of 32,441 appendix samples and 2 out of 14,692 samples, respectively, indicating a surprisingly high prevalence abnormal PrP. Given the 178 vCJD cases hitherto reported in the UK, there appears to be a discordance between the population prevalence data and the number of observed clinical cases, which challenges the assumption of a perfect association

between PrP deposition in LRS tissues and vCJD disease. To provide insight into this discordance, we aim to develop a classifier based on epigenomic profiles that distinguishes prion infection from healthy controls.

**Materials and Methods:** A unique archive of fixed and frozen LRS tissues including tonsil and appendix from vCJD patients, blood and brain samples from a range of human prion disease patients. Samples have been age/ sex-matched to controls not diagnosed with neurodegenerative disease. For DNA methylation profiling, extracted genomic DNA has been subjected to bisulfite conversion and, subsequently, hybridized to the Illumina 450K or 850K EPIC arrays. Training of a deep learning neural network model was performed using these samples.

**Results:** Initial results explored the diagnostic potential of an Illumina 450K dataset using deep learning neural network classifier. This DNA methylation study was conducted using whole bloods from 106 healthy controls and 114 sCJD patients (age/ sex-matched). Using data from the 1000 most significantly altered methylation loci, this data was randomized and partitioned into training and test set of 1:1 case-control ratio. After 10-fold validation, the model had an accuracy of 85.64% (± 1.14%) with an upper limit of 90.00% accuracy. The trained neural network model demonstrated a better performance compared to a basic Random Forest classifier with an AUC of 0.965 and 0.841, respectively.

**Conclusions:** As a proof of concept, we have developed an accurate classification tool using relatively small numbers of differentially methylated loci in blood. We are extending these results to a wider panel of neurodegenerative dementias and LRS tissues.

**KEYWORDS:** Epigenetics; EWAS; DNA methylation; Variant Creutzfeldt-Jakob; Machine learning; Appendix; Tonsil; Lymphoreticular tissue

## 201. TDP-43 induces SOD1 aggregation and toxicity via tryptophan interactions that present a novel drug target

Michèle G. DuValª, Edward Pokrishevskyᵇ, Vijaya K. Hingeᶜ,>d, Jenna Bratvoldª, Richard Kanyoª,ᵉ, Natalie Snyderª, Nikolay Blinovᶜ,>d, Andriy Kovalenkoᶜ,>d, Neil R. Cashmanᵇ and W. Ted Allisonª,>e,f

<footnote>ªDepartment of Biological Sciences, University of Alberta, Edmonton, AB, Canada; ᵇDjavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, BC, Canada; ᶜNational Institute for Nanotechnology, Edmonton, AB, Canada; ᵈDepartment of Mechanical Engineering, University of Alberta, Edmonton, AB, Canada; ᵉCentre for Prions and Protein Folding Diseases, University</footnote>
<figure><img src="image_123.png" /></figure>

of Alberta, Edmonton, AB, Canada; Department of Medical Genetics, University of Alberta, Edmonton, AB, Canada

CONTACT Michèle G. DuVal mgduval@ualberta.ca

## ABSTRACT

Amyotrophic lateral sclerosis (ALS) is a devastating motor neurodegenerative disease which features a diversity of protein inclusions. Aggregates of superoxide dismutase 1 (SOD1) or of TAR-DNA binding protein-43 (TDP-43) have been identified in familial and sporadic cases. Misfolded wildtype SOD1 can spread in a prion-like fashion, evidenced by induction of wildtype SOD1 misfolding by mutant SOD1 or by TDP-43 variants, as observed in cells and cultured neurons [1-3]. The sole tryptophan in SOD1 at position 32 (W32) is instrumental in SOD1 misfolding [1,2,4], and we hypothesize that TDP-43 also induces SOD1 misfolding via interactions between their respective tryptophan residues. We measured the contribution of these tryptophans to SOD1 aggregation *in vitro* and toxicity *in vivo* by expressing variants with combinations of tryptophan substitutions.

In HEK293 cells, TDP-43 lacking all six tryptophans induced little SOD1 inclusion formation, which was replicated with TDP-43 variants lacking two specific tryptophans. In a zebrafish model of motor neuron disease [5-7], disturbed motor axon morphology caused by TDP-43 and SOD1 co-expression was similarly rescued, with congruent results for substitutions of specific tryptophans in TDP-43. Thus TDP-43 can induce SOD1 aggregation and toxicity via interactions between tryptophans on the two proteins, which may occur under pathological conditions and thereby advance disease. Similarly, mutating W32 in the human SOD1 delivered to zebrafish lead to significant reduction of motor neuron phenotypes (vs wildtype SOD1), including preservation of axon morphology and of motor output (swimming).

Candidate therapeutic compound 5'-fluorouridine, known to bind at the W32 region of SOD1 [8], successfully rescued cross-seeding of SOD1 by TDP-43 and subsequent motor neuron toxicity. Next, to identify additional compounds that would bind at W32, high throughput virtual and pharmacophore screening was performed with modelling of the W32 binding site and screening of FDA-approved drugs that closely match key features of 5'-fluorouridine. From among top-ranked compounds, telbivudine significantly rescued SOD1-induced motor neuron phenotypes [9]. In conclusion, we found that tryptophan 32 in SOD1 is a significant contributor to SOD1 aggregation and toxicity, including via

novel TDP-43-SOD1 interactions. These interactions represent new pharmacologic targets and we identified FDA-approved compounds, predicted to interact with SOD1 at W32, that are effective in preventing inclusion formation and toxicity.

**KEYWORDS:** SOD1; TDP-43; prion-like; ALS; tryptophan; 5'-fluorouridine; telbivudine; zebrafish

## References

[1] Grad et al., PNAS. 2011;111:3620-3625.

[2] Taylor et al., J Biol Chem. 2007;282:16329-16335.

[3] Pokrishevsky et al., Sci Rep. 2016;6:22155.

[4] Pokrishevsky et al., Sci Rep. 2018; 8:15590.

[5] Sakowski et al., Mol Neurodegener. 2012;7:44.

[6] DuVal et al., PLoS One. 2014;9:e89183

[7] Edens et al., eLife. 2017; 6:e25453

[8] Wright et al., Nat Commun. 2013;4:1758

[9] DuVal et al., Neurobiol Dis 2019; 124: 297-310

## 202. Competitive ELISAs to determine PrP<sup>Sc</sup> content in crude homogenates without the need for denaturation or protease treatments

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

<sup>a</sup>Centre for Prions and Protein Folding Diseases, University of Alberta; <sup>b</sup>Department of Biochemistry; <sup>c</sup>Department of Medicine; <sup>d</sup>Department of Biological Sciences; <sup>e</sup>Department of Agricultural, Food, and Nutritional Science, Edmonton, Alberta, Canada

CONTACT Xinli Tang xinli@ualberta.ca

## ABSTRACT

**Background:** The lack of readily accessible, PrP<sup>Sc</sup>-specific monoclonal antibodies has hampered the detection of PrP<sup>Sc</sup> in brain homogenates and other samples. The presence of PrP<sup>C</sup> and other, non-infectious PrP conformers confounds diagnostic assays that lack specificity for native PrP<sup>Sc</sup>. Currently used diagnostics often rely on the partial protease resistance of PrP<sup>Sc</sup>, which is used to differentiate PrP<sup>Sc</sup> from PrP<sup>C</sup> and other PrP conformers. Furthermore, the aggregated nature of PrP<sup>Sc</sup> obscures many epitopes, making denaturation of PrP<sup>Sc</sup> a prerequisite for many detection assays. Together these requirements affect the specificity and sensitivity of the detection assays and increase the necessary labour, time, and resources to determine if a sample contains PrP<sup>Sc</sup>.

**Materials and Methods:** We immunized mice with a structural and immunological mimic for PrP<sup>Sc</sup>, which was based on the fungal HET-s prion domain as a scaffold. At the end
<figure><img src="image_122.png" /></figure>

of Alberta, Edmonton, AB, Canada; Department of Medical Genetics, University of Alberta, Edmonton, AB, Canada

CONTACT Michèle G. DuVal mgduval@ualberta.ca

## ABSTRACT

Amyotrophic lateral sclerosis (ALS) is a devastating motor neurodegenerative disease which features a diversity of protein inclusions. Aggregates of superoxide dismutase 1 (SOD1) or of TAR-DNA binding protein-43 (TDP-43) have been identified in familial and sporadic cases. Misfolded wildtype SOD1 can spread in a prion-like fashion, evidenced by induction of wildtype SOD1 misfolding by mutant SOD1 or by TDP-43 variants, as observed in cells and cultured neurons [1-3]. The sole tryptophan in SOD1 at position 32 (W32) is instrumental in SOD1 misfolding [1,2,4], and we hypothesize that TDP-43 also induces SOD1 misfolding via interactions between their respective tryptophan residues. We measured the contribution of these tryptophans to SOD1 aggregation *in vitro* and toxicity *in vivo* by expressing variants with combinations of tryptophan substitutions.

In HEK293 cells, TDP-43 lacking all six tryptophans induced little SOD1 inclusion formation, which was replicated with TDP-43 variants lacking two specific tryptophans. In a zebrafish model of motor neuron disease [5-7], disturbed motor axon morphology caused by TDP-43 and SOD1 co-expression was similarly rescued, with congruent results for substitutions of specific tryptophans in TDP-43. Thus TDP-43 can induce SOD1 aggregation and toxicity via interactions between tryptophans on the two proteins, which may occur under pathological conditions and thereby advance disease. Similarly, mutating W32 in the human SOD1 delivered to zebrafish lead to significant reduction of motor neuron phenotypes (vs wildtype SOD1), including preservation of axon morphology and of motor output (swimming).

Candidate therapeutic compound 5'-fluorouridine, known to bind at the W32 region of SOD1 [8], successfully rescued cross-seeding of SOD1 by TDP-43 and subsequent motor neuron toxicity. Next, to identify additional compounds that would bind at W32, high throughput virtual and pharmacophore screening was performed with modelling of the W32 binding site and screening of FDA-approved drugs that closely match key features of 5'-fluorouridine. From among top-ranked compounds, telbivudine significantly rescued SOD1-induced motor neuron phenotypes [9]. In conclusion, we found that tryptophan 32 in SOD1 is a significant contributor to SOD1 aggregation and toxicity, including via

novel TDP-43-SOD1 interactions. These interactions represent new pharmacologic targets and we identified FDA-approved compounds, predicted to interact with SOD1 at W32, that are effective in preventing inclusion formation and toxicity.

**KEYWORDS:** SOD1; TDP-43; prion-like; ALS; tryptophan; 5'-fluorouridine; telbivudine; zebrafish

## References

[1] Grad et al., PNAS. 2011;111:3620-3625.

[2] Taylor et al., J Biol Chem. 2007;282:16329-16335.

[3] Pokrishevsky et al., Sci Rep. 2016;6:22155.

[4] Pokrishevsky et al., Sci Rep. 2018; 8:15590.

[5] Sakowski et al., Mol Neurodegener. 2012;7:44.

[6] DuVal et al., PLoS One. 2014;9:e89183

[7] Edens et al., eLife. 2017; 6:e25453

[8] Wright et al., Nat Commun. 2013;4:1758

[9] DuVal et al., Neurobiol Dis 2019; 124: 297-310

## 202. Competitive ELISAs to determine PrP<sup>Sc</sup> content in crude homogenates without the need for denaturation or protease treatments

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

<sup>a</sup>Centre for Prions and Protein Folding Diseases, University of Alberta; <sup>b</sup>Department of Biochemistry; <sup>c</sup>Department of Medicine; <sup>d</sup>Department of Biological Sciences; <sup>e</sup>Department of Agricultural, Food, and Nutritional Science, Edmonton, Alberta, Canada

CONTACT Xinli Tang xinli@ualberta.ca

## ABSTRACT

**Background:** The lack of readily accessible, PrP<sup>Sc</sup>-specific monoclonal antibodies has hampered the detection of PrP<sup>Sc</sup> in brain homogenates and other samples. The presence of PrP<sup>C</sup> and other, non-infectious PrP conformers confounds diagnostic assays that lack specificity for native PrP<sup>Sc</sup>. Currently used diagnostics often rely on the partial protease resistance of PrP<sup>Sc</sup>, which is used to differentiate PrP<sup>Sc</sup> from PrP<sup>C</sup> and other PrP conformers. Furthermore, the aggregated nature of PrP<sup>Sc</sup> obscures many epitopes, making denaturation of PrP<sup>Sc</sup> a prerequisite for many detection assays. Together these requirements affect the specificity and sensitivity of the detection assays and increase the necessary labour, time, and resources to determine if a sample contains PrP<sup>Sc</sup>.

**Materials and Methods:** We immunized mice with a structural and immunological mimic for PrP<sup>Sc</sup>, which was based on the fungal HET-s prion domain as a scaffold. At the end
of the immunization schedule we used their spleens to generate hybridomas for the production of monoclonal antibodies. Here, we tested the resulting antibodies in conventional and competitive ELISAs for their ability to detect native $PrP^{Sc}$ from different prion isolates and to distinguish $PrP^{Sc}$ from $PrP^{C}$ and other PrP conformers.

**Results:** Immunization with our newly developed, structural mimic gave rise to a $PrP^{Sc}$-specific immune response in 8/8 mice. The resulting monoclonal antibodies (IgGs and IgMs) recognized native $PrP^{Sc}$ in crude brain homogenates without any pre-treatment, but did not bind to $PrP^{C}$, recombinant PrP, denatured PrP, unseeded PrP amyloid, or any linear peptide of PrP. We tested a variety of assay formats and obtained the most reliable results using a competitive/inhibition ELISA. Here, our original antigen was used to coat the assay plates and to compete with $PrP^{Sc}$ in the samples (typically crude brain homogenate) for binding of unconjugated antibodies. A conventional detection system using a secondary antibody (HRP-goat-anti-mouse IgG or IgM) generated the final colorimetric readout. The assay recognized native $PrP^{Sc}$ from all prion isolates tested, including CWD, BSE (C-, H-, and L-type), scrapie (RML), TME (Hyper and Drowsy), sCJD, vCJD, fCJD, FFI, and GSS, indicating its ability to detect a widely-shared $PrP^{Sc}$-specific epitope.

**Conclusions:** Our new, $PrP^{Sc}$-specific monoclonal antibodies allowed the development of a competitive ELISA for the detection of native $PrP^{Sc}$ without protease or denaturant pretreatment. Furthermore, the broad specificity of these antibodies opens the possibility to develop a widely applicable and sensitive diagnostic tool for the direct detection of $PrP^{Sc}$.

## 203. Seeding of a fast acting Aß peptide accelerates AD-like pathology in APPNL-G-F mouse model with no impairment in learning and memory

Sean G. Lacoursiere, Majid H. Mohajerani and Robert J. Sutherland

Canadian Centre for Behavioural Neuroscience, University of Lethbridge

CONTACT Sean G. Lacoursiere sean.lacoursiere@uleth.ca

### ABSTRACT

Alzheimer's disease (AD) is a neurodegenerative dementia characterized by the stereotypical conformational misfolding of proteins. Of specific interest is amyloid-β (Aβ) due to its prion-like properties evidenced by the seeding of Aβ accelerating plaque deposition in brain. However, the role of Aβ in AD remains unclear. Many hypothesize that increasing Aβ impairs cognition; and therefore, accelerating Aβ

pathology should increase the extent of cognitive decline to the point of dementia. We performed bilateral intracerebral microinjections of a fast-acting Aβ peptide into the medial entorhinal cortex of the knock-in APPNL-G-F mouse model of AD at 2 months of age. We found deposition of Aβ plaque occurred sooner and to a greater extent compared to controls but without obvious impairment in cognition or overall health. We found that at 6 months the APPNL-G-F mice showed similar memory performance in a no-platform Morris Water Task probe test compared to controls. Immunohistochemical labelling showed dramatically increased 82E1 and Iba1 label in these mice 1 month following injection, indicating increased Aβ and microgliosis pathology, respectively. Our results replicate previous reports of the seeding properties of Aβ and its prion-like behaviour. They also support notions that Aβ may not be sufficient to cause cognitive decline in Alzheimer's disease. The Aβ pathology in AD may be a response to some other trigger. The seeding properties of Aβ may be an adaptive response to control some other process.

## 204. Alteration to the prion protein's central region sequence retains structure but results in toxicity

Graham P. Roseman<sup>a</sup>, Bei Wu<sup>b</sup>, Mark Wadolkowski<sup>a</sup>, David A. Harris<sup>b</sup> and Glenn L. Millhauser<sup>a</sup>

<sup>a</sup>University of California Santa Cruz; <sup>b</sup>Boston University School of Medicine

CONTACT Graham P. Roseman groseman@ucsc.edu

### ABSTRACT

The central region (CR) of the prion protein ($PrP^{C}$) is one of the most highly conserved sequences across species. Deletion of the entire prion protein gene in mice is fairly tolerated, but deletion of just the CR (ΔCR: 105–125) causes neonatal fatality in mice [1] and toxicity in cell culture as seen by spontaneous currents using patch clamp electrophysiology experiments. There is a great interest in understanding the toxicity of this deletion mutant because the neurotoxicity has similarities to that of classic prion diseases except without the aggregation of $PrP^{C}$. Biophysical studies have shown that ΔCR causes two main changes: weakening of the metal-driven cis interaction between the N- and C- terminal domains [2] and deletion of the region of the protein where regulatory proteolysis occurs, termed α-cleavage. What is not known is whether the neurotoxicity that ΔCR causes is a result of a weakened cis interaction or elimination of
<figure><img src="image_123.png" /></figure>

of the immunization schedule we used their spleens to generate hybridomas for the production of monoclonal antibodies. Here, we tested the resulting antibodies in conventional and competitive ELISAs for their ability to detect native $PrP^{Sc}$ from different prion isolates and to distinguish $PrP^{Sc}$ from $PrP^{C}$ and other PrP conformers.

**Results:** Immunization with our newly developed, structural mimic gave rise to a $PrP^{Sc}$-specific immune response in 8/8 mice. The resulting monoclonal antibodies (IgGs and IgMs) recognized native $PrP^{Sc}$ in crude brain homogenates without any pre-treatment, but did not bind to $PrP^{C}$, recombinant PrP, denatured PrP, unseeded PrP amyloid, or any linear peptide of PrP. We tested a variety of assay formats and obtained the most reliable results using a competitive/inhibition ELISA. Here, our original antigen was used to coat the assay plates and to compete with $PrP^{Sc}$ in the samples (typically crude brain homogenate) for binding of unconjugated antibodies. A conventional detection system using a secondary antibody (HRP-goat-anti-mouse IgG or IgM) generated the final colorimetric readout. The assay recognized native $PrP^{Sc}$ from all prion isolates tested, including CWD, BSE (C-, H-, and L-type), scrapie (RML), TME (Hyper and Drowsy), sCJD, vCJD, fCJD, FFI, and GSS, indicating its ability to detect a widely-shared $PrP^{Sc}$-specific epitope.

**Conclusions:** Our new, $PrP^{Sc}$-specific monoclonal antibodies allowed the development of a competitive ELISA for the detection of native $PrP^{Sc}$ without protease or denaturant pretreatment. Furthermore, the broad specificity of these antibodies opens the possibility to develop a widely applicable and sensitive diagnostic tool for the direct detection of $PrP^{Sc}$.

## 203. Seeding of a fast acting Aß peptide accelerates AD-like pathology in APPNL-G-F mouse model with no impairment in learning and memory

Sean G. Lacoursiere, Majid H. Mohajerani and Robert J. Sutherland

Canadian Centre for Behavioural Neuroscience, University of Lethbridge

CONTACT Sean G. Lacoursiere sean.lacoursiere@uleth.ca

### ABSTRACT

Alzheimer's disease (AD) is a neurodegenerative dementia characterized by the stereotypical conformational misfolding of proteins. Of specific interest is amyloid-β (Aβ) due to its prion-like properties evidenced by the seeding of Aβ accelerating plaque deposition in brain. However, the role of Aβ in AD remains unclear. Many hypothesize that increasing Aβ impairs cognition; and therefore, accelerating Aβ

pathology should increase the extent of cognitive decline to the point of dementia. We performed bilateral intracerebral microinjections of a fast-acting Aβ peptide into the medial entorhinal cortex of the knock-in APPNL-G-F mouse model of AD at 2 months of age. We found deposition of Aβ plaque occurred sooner and to a greater extent compared to controls but without obvious impairment in cognition or overall health. We found that at 6 months the APPNL-G-F mice showed similar memory performance in a no-platform Morris Water Task probe test compared to controls. Immunohistochemical labelling showed dramatically increased 82E1 and Iba1 label in these mice 1 month following injection, indicating increased Aβ and microgliosis pathology, respectively. Our results replicate previous reports of the seeding properties of Aβ and its prion-like behaviour. They also support notions that Aβ may not be sufficient to cause cognitive decline in Alzheimer's disease. The Aβ pathology in AD may be a response to some other trigger. The seeding properties of Aβ may be an adaptive response to control some other process.

## 204. Alteration to the prion protein's central region sequence retains structure but results in toxicity

Graham P. Roseman<sup>a</sup>, Bei Wu<sup>b</sup>, Mark Wadolkowski<sup>a</sup>, David A. Harris<sup>b</sup> and Glenn L. Millhauser<sup>a</sup>

<sup>a</sup>University of California Santa Cruz; <sup>b</sup>Boston University School of Medicine

CONTACT Graham P. Roseman groseman@ucsc.edu

### ABSTRACT

The central region (CR) of the prion protein ($PrP^C$) is one of the most highly conserved sequences across species. Deletion of the entire prion protein gene in mice is fairly tolerated, but deletion of just the CR (ΔCR: 105–125) causes neonatal fatality in mice [1] and toxicity in cell culture as seen by spontaneous currents using patch clamp electrophysiology experiments. There is a great interest in understanding the toxicity of this deletion mutant because the neurotoxicity has similarities to that of classic prion diseases except without the aggregation of $PrP^C$. Biophysical studies have shown that ΔCR causes two main changes: weakening of the metal-driven cis interaction between the N- and C- terminal domains [2] and deletion of the region of the protein where regulatory proteolysis occurs, termed α-cleavage. What is not known is whether the neurotoxicity that ΔCR causes is a result of a weakened cis interaction or elimination of
<figure><img src="image_124.png" /></figure>

proteolytic sites. This study aims to ask that question by using a designed PrP<sup>C</sup> construct that replaces the central region with a flexible glycine-serine (GS) rich linker that retains the metal-driven *cis* interaction of wild type PrP<sup>C</sup> while blocking the cleavability of this region. Results from the GS linker show that the *cis* interaction is retained as tested by NMR, α-cleavage was blocked *in vitro* and in cell culture, and toxicity was seen using electrophysiological experiments. To test if the toxicity generated from the GS linker construct was due from eliminating α-cleavage, a rescue construct was designed by reintroducing the canonical α-cleavage sequence into the GS linker backbone. α-cleavage was successfully reintroduced and the metal-driven *cis* interaction was retained. However, when this construct was tested using the electrophysiology experiments, spontaneous currents still persisted, indicating that there was still toxicity. Overall these results implicate that the sequence of the central region is extremely important for the structure and function of the prion protein, and when deleted or mutated, spontaneous neurotoxicity occurs.

**KEYWORDS:** Metal binding; copper; cleavage; NMR; electrophysiology

## References

[1] Li et al., EMBO. 2007;26:548-558.

[2] Wu et al., eLife. 2017;6:e23,473

## **205. Deep learning-based pre-mortem diagnostic methods for sporadic Creutzfeldt-Jakob disease using multiple CSF biomarkers**

Sol Moe Lee<sup>a,b</sup>, Jae Wook Hyeon<sup>a</sup>, Soo-Jin Kim<sup>b</sup>, Heebal Kim<sup>b</sup>, Ran Noh<sup>a</sup>, Seonghan Kim<sup>a</sup>, Yeong Seon Lee<sup>a</sup>, Sung Soon Kim<sup>a</sup> and Su Yeon Kim<sup>a</sup>

CONTACT Sol Moe Lee arabe@korea.kr; Su Yeon Kim tenksy@korea.kr

## ABSTRACT

Creutzfeldt-Jakob disease (CJD) is a fatal neurodegenerative disorder which is characterized by the abnormal accumulation of misfolded prion protein (PrP<sup>Sc</sup>)

affecting the central nervous system. The diagnosis of CJD can only be confirmed by abnormal protease-resistant prion protein accumulation in post-mortem brain tissue. Sporadic CJD (sCJD) is the most common subtype, and the relationships between sCJD and cerebrospinal fluid (CSF) proteins such as 14-3-3, tau, and α-synuclein (a-syn) have been investigated for their potential values in pre-mortem diagnosis. Deep learning (DL), a machine learning (ML) based on neural networks, has provided a powerful remedy for analysing large-scale data in diverse fields such as computer vision, natural language processing, and signal modelling for several years. Recently, it is reported that DL has attracted large attention in neurodegenerative disease research as well. Here, ML and DL analysis were performed using multiple CSF biomarkers related with neuronal diseases including CJD and Alzheimer's diseases. We performed ML analysis including decision trees and SVMs using WEKA (Waikato Environment for Knowledge Analysis 3.8.2), and DL analysis was conducted with Keras neural network library. Enzyme-linked immunosorbent assays were performed on phospho-tau (p-tau), total-tau (t-tau), a-syn, and β-amyloid (1-42), and western blot analysis was performed for 14-3-3 protein from CSF samples of 49 sCJD and 256 non-CJD Korean patients, respectively. The best performing ML model demonstrated 78.85% accuracy. The deep neural network structure which showed the best DL performance was comprised of one input, five hidden, and one output layers, with 20, 40, 30, 20, and 12 hidden unit numbers per hidden layer, respectively. For the analysed CSF biomarkers, the DL model demonstrated 90.38% accuracy, 83.33% sensitivity, and 92.5% specificity for the three-protein combination of t-tau, p-tau, and a-syn. DL-aided pre-mortem diagnosis may provide a suitable tool for discriminating CJD patients from non-CJD patients. For further study, we plan to perform DL analysis using stacked sample sizes via ELISA and western blot analysis to establish a DL model with greater high performance and classification accuracy.

## **206. Characterizing the molecular environment of the bank vole prion protein using mass spectrometry**

Hamza Arshad<sup>a,b</sup>, Declan Williams<sup>a</sup>, Lech Kaczmarczyk<sup>c</sup>, Walker Jackson<sup>c</sup>, Gerold Schmitt-Ulms<sup>a,d</sup>, Joel Watts<sup>a,b</sup>

<footnote><sup>a</sup>Division of Bacterial Disease Research, Center for Infectious Diseases Research, Korea National Institute of Health, Centers for Disease Control and Prevention, Cheongju-si, Chungcheongbuk-do, South Korea; <sup>b</sup>Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, South Korea</footnote>

<footnote><sup>a</sup>Tanz Centre for Research in Neurodegenerative Diseases, Toronto, Canada; <sup>b</sup>Department of Biochemistry, University of Toronto, Toronto, Canada; <sup>c</sup>Wallenberg Center for Molecular Medicine,</footnote>
<figure><img src="image_124.png" /></figure>

proteolytic sites. This study aims to ask that question by using a designed PrP<sup>C</sup> construct that replaces the central region with a flexible glycine-serine (GS) rich linker that retains the metal-driven *cis* interaction of wild type PrP<sup>C</sup> while blocking the cleavability of this region. Results from the GS linker show that the *cis* interaction is retained as tested by NMR, α-cleavage was blocked *in vitro* and in cell culture, and toxicity was seen using electrophysiological experiments. To test if the toxicity generated from the GS linker construct was due from eliminating α-cleavage, a rescue construct was designed by reintroducing the canonical α-cleavage sequence into the GS linker backbone. α-cleavage was successfully reintroduced and the metal-driven *cis* interaction was retained. However, when this construct was tested using the electrophysiology experiments, spontaneous currents still persisted, indicating that there was still toxicity. Overall these results implicate that the sequence of the central region is extremely important for the structure and function of the prion protein, and when deleted or mutated, spontaneous neurotoxicity occurs.

**KEYWORDS:** Metal binding; copper; cleavage; NMR; electrophysiology

## References

[1] Li et al., EMBO. 2007;26:548-558.

[2] Wu et al., eLife. 2017;6:e23,473

## **205. Deep learning-based pre-mortem diagnostic methods for sporadic Creutzfeldt-Jakob disease using multiple CSF biomarkers**

Sol Moe Lee<sup>a,b</sup>, Jae Wook Hyeon<sup>a</sup>, Soo-Jin Kim<sup>b</sup>, Heebal Kim<sup>b</sup>, Ran Noh<sup>a</sup>, Seonghan Kim<sup>a</sup>, Yeong Seon Lee<sup>a</sup>, Sung Soon Kim<sup>a</sup> and Su Yeon Kim<sup>a</sup>

CONTACT Sol Moe Lee arabe@korea.kr; Su Yeon Kim tenksy@korea.kr

## ABSTRACT

Creutzfeldt-Jakob disease (CJD) is a fatal neurodegenerative disorder which is characterized by the abnormal accumulation of misfolded prion protein (PrP<sup>Sc</sup>)

affecting the central nervous system. The diagnosis of CJD can only be confirmed by abnormal protease-resistant prion protein accumulation in post-mortem brain tissue. Sporadic CJD (sCJD) is the most common subtype, and the relationships between sCJD and cerebrospinal fluid (CSF) proteins such as 14-3-3, tau, and α-synuclein (a-syn) have been investigated for their potential values in pre-mortem diagnosis. Deep learning (DL), a machine learning (ML) based on neural networks, has provided a powerful remedy for analysing large-scale data in diverse fields such as computer vision, natural language processing, and signal modelling for several years. Recently, it is reported that DL has attracted large attention in neurodegenerative disease research as well. Here, ML and DL analysis were performed using multiple CSF biomarkers related with neuronal diseases including CJD and Alzheimer's diseases. We performed ML analysis including decision trees and SVMs using WEKA (Waikato Environment for Knowledge Analysis 3.8.2), and DL analysis was conducted with Keras neural network library. Enzyme-linked immunosorbent assays were performed on phospho-tau (p-tau), total-tau (t-tau), a-syn, and β-amyloid (1-42), and western blot analysis was performed for 14-3-3 protein from CSF samples of 49 sCJD and 256 non-CJD Korean patients, respectively. The best performing ML model demonstrated 78.85% accuracy. The deep neural network structure which showed the best DL performance was comprised of one input, five hidden, and one output layers, with 20, 40, 30, 20, and 12 hidden unit numbers per hidden layer, respectively. For the analysed CSF biomarkers, the DL model demonstrated 90.38% accuracy, 83.33% sensitivity, and 92.5% specificity for the three-protein combination of t-tau, p-tau, and a-syn. DL-aided pre-mortem diagnosis may provide a suitable tool for discriminating CJD patients from non-CJD patients. For further study, we plan to perform DL analysis using stacked sample sizes via ELISA and western blot analysis to establish a DL model with greater high performance and classification accuracy.

## **206. Characterizing the molecular environment of the bank vole prion protein using mass spectrometry**

Hamza Arshad<sup>a,b</sup>, Declan Williams<sup>a</sup>, Lech Kaczmarczyk<sup>c</sup>, Walker Jackson<sup>c</sup>, Gerold Schmitt-Ulms<sup>a,d</sup>, Joel Watts<sup>a,b</sup>

<footnote><sup>a</sup>Division of Bacterial Disease Research, Center for Infectious Diseases Research, Korea National Institute of Health, Centers for Disease Control and Prevention, Cheongju-si, Chungcheongbuk-do, South Korea; <sup>b</sup>Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, South Korea</footnote>

<footnote><sup>a</sup>Tanz Centre for Research in Neurodegenerative Diseases, Toronto, Canada; <sup>b</sup>Department of Biochemistry, University of Toronto, Toronto, Canada; <sup>c</sup>Wallenberg Center for Molecular Medicine,</footnote>
Linköping University, Linköping, Sweden;Department of
Laboratory Medicine and Pathobiology, University of Toronto,
Toronto, Canada

CONTACT Hamza Arshad hamza.arshad@mail.utoronto.ca

ABSTRACT

**Background:** The bank vole prion protein (BVPrP) is able to replicate a wide range of prion strains from different species and has been characterized as a universal acceptor of prions [1,2]. The sequence of mature BVPrP differs from that of mouse PrP (MoPrP) at only eight different amino acid positions. We hypothesized that these unique residues may allow BVPrP to tailor its molecular environment through novel protein-protein interactions. To address this issue, we generated knock-in (ki) mice that express BVPrP (M109 isoform) at physiological levels in the brain. The PrP interactomes in ki mice expressing BVPrP and wild-type mice expressing MoPrP were compared using quantitative mass spectrometry.

**Materials and Methods:** BVPrP ki mice, wild-type C57Bl/6 mice, and PrP knockout mice were subjected to time-controlled transcardiac perfusion crosslinking (tcTPC) and then PrP- containing protein complexes were isolated from the brain by immunoprecipitation [3]. Triplicate samples were analysed by quantitative mass spectrometry to permit a direct comparison of the interactomes of MoPrP and BVPrP. The interac-tome analysis was conducted twice, using two different anti-PrP antibodies recognizing distinct epitopes, to ensure that all relevant interactions were captured.

**Results:** The molecular environment of BVPrP was very similar to that of MoPrP. Many known MoPrP interactors were found in our experiments, including NCAM, 4F2, basigin, as well as subtle interactors such as ZIP6 and ZIP10. We found that there was significant overlap between the top interactors found in the BVPrP and MoPrP samples. However, we also identified sev-eral proteins that were reproducibly enriched in the samples derived from BVPrP ki mice. These hits are currently being validated using cultured cell models.

**Conclusions:** We conclude that the molecular environ-ments of BVPrP and MoPrP in the brains of mice are very similar. Nonetheless, several proteins appear to preferentially interact with BVPrP. These proteins may help to explain the unique behaviour of BVPrP.

**KEYWORDS:** Bank vole; mass spectrometry; interac-tome; species barrier

References

[1] Watts et al., PLoS Pathog. 2014;10(4):e1003990.

[2] Agrimi et al., PLoS Pathog. 2008;4(7):e1000113.

[3] Schmitt-Ulms et al., Nat Biotech. 2004;22(6):724-731.

**207. Neurodegeneration in the enteric nervous system in an MPTP induced model of Parkinson's disease**

Laura J Ellett<sup>a,b,c</sup> David I Finkelstein<sup>c</sup> and Victoria A. Lawson<sup>a,b,c</sup>

<sup>a</sup>The Department of Pathology; <sup>b</sup>The Department of Microbiology and Immunology; <sup>c</sup>The Florey Institute for Neuroscience and Mental Health, The University of Melbourne, Victoria, Australia

CONTACT Victoria A. Lawson vlawson@unimelb.edu.au

ABSTRACT

The spread of α-synuclein pathology in Parkinson's disease has been described as prion like with pathology thought to spread from the peripheral to central nervous system in at least some forms of the disease. Prion pathology and gastrointestinal dysfunction has also been reported in some forms of prion disease.

Following MPTP-lesioning, a well characterized model of Parkinson's disease, C57BL/6 mice develop a constipation phenotype characterized by reduced stool frequency. We have recently correlated this dysfunction with enteric glial cell reactivity and the loss of specific neuronal populations in the enteric nervous system [1]. In the present study we investigated the mechanism of MPTP induced neurodegeneration in the enteric nervous system.

MPTP induced toxicity is dependent on the uptake of its toxic metabolite MPP+ by dopamine active transporter (DAT) expressed on dopaminergic neurons. DAT expression is highest in the ileum of the gastrointestinal tract. Consistent with this tyrosine hydroxylase (TH) immunoreactive (IR) dopaminergic neurons were significantly reduced in the ileum of MPTP-lesioned mice, but unaffected in the colon where DAT expression is lower.

Although MPTP-lesioning is not generally associated with Lewy pathology, MPTP-lesioning led to a change in localization of α-synuclein in DAT-IR neurons of the ileum. MPTP treatment did not affect α-synuclein localization in the colon.

Non-dopaminergic neurons identified by the expression of neurofilament (NFM-IR neurons) were also reduced in the myenteric plexus of the ileum, but not colon of MPTP lesioned mice.

We hypothesize that MPTP-initiated loss of non-dopaminergic cell populations may in response to MPTP induced changes in α-synuclein and/or the response of enteric glial cells to MPTP induced injury. This hypothesis is consistent with a toxic insult triggering Parkinson's disease pathology in a vulnerable cell population within the gastrointestinal tract, with secondary neurodegeneration
Linköping University, Linköping, Sweden; Department of
Laboratory Medicine and Pathobiology, University of Toronto,
Toronto, Canada

CONTACT Hamza Arshad hamza.arshad@mail.utoronto.ca

ABSTRACT

**Background:** The bank vole prion protein (BVPrP) is able to replicate a wide range of prion strains from different species and has been characterized as a universal acceptor of prions [1,2]. The sequence of mature BVPrP differs from that of mouse PrP (MoPrP) at only eight different amino acid positions. We hypothesized that these unique residues may allow BVPrP to tailor its molecular environment through novel protein-protein interactions. To address this issue, we generated knock-in (ki) mice that express BVPrP (M109 isoform) at physiological levels in the brain. The PrP interactomes in ki mice expressing BVPrP and wild-type mice expressing MoPrP were compared using quantitative mass spectrometry.

**Materials and Methods:** BVPrP ki mice, wild-type C57Bl/6 mice, and PrP knockout mice were subjected to time-controlled transcardiac perfusion crosslinking (tcTPC) and then PrP-containing protein complexes were isolated from the brain by immunoprecipitation [3]. Triplicate samples were analysed by quantitative mass spectrometry to permit a direct comparison of the interactomes of MoPrP and BVPrP. The interactome analysis was conducted twice, using two different anti-PrP antibodies recognizing distinct epitopes, to ensure that all relevant interactions were captured.

**Results:** The molecular environment of BVPrP was very similar to that of MoPrP. Many known MoPrP interactors were found in our experiments, including NCAM, 4F2, basigin, as well as subtle interactors such as ZIP6 and ZIP10. We found that there was significant overlap between the top interactors found in the BVPrP and MoPrP samples. However, we also identified several proteins that were reproducibly enriched in the samples derived from BVPrP ki mice. These hits are currently being validated using cultured cell models.

**Conclusions:** We conclude that the molecular environments of BVPrP and MoPrP in the brains of mice are very similar. Nonetheless, several proteins appear to preferentially interact with BVPrP. These proteins may help to explain the unique behaviour of BVPrP.

**KEYWORDS:** Bank vole; mass spectrometry; interactome; species barrier

References

[1] Watts et al., PLoS Pathog. 2014;10(4):e1003990.

[2] Agrimi et al., PLoS Pathog. 2008;4(7):e1000113.

[3] Schmitt-Ulms et al., Nat Biotech. 2004;22(6):724-731.

**207. Neurodegeneration in the enteric nervous system in an MPTP induced model of Parkinson's disease**

Laura J Ellett<sup>a,b,c</sup> David I Finkelstein<sup>c</sup> and Victoria A. Lawson<sup>a,b,c</sup>

<sup>a</sup>The Department of Pathology; <sup>b</sup>The Department of Microbiology and Immunology; <sup>c</sup>The Florey Institute for Neuroscience and Mental Health, The University of Melbourne, Victoria, Australia

CONTACT Victoria A. Lawson vlawson@unimelb.edu.au

ABSTRACT

The spread of α-synuclein pathology in Parkinson's disease has been described as prion like with pathology thought to spread from the peripheral to central nervous system in at least some forms of the disease. Prion pathology and gastrointestinal dysfunction has also been reported in some forms of prion disease.

Following MPTP-lesioning, a well characterized model of Parkinson's disease, C57BL/6 mice develop a constipation phenotype characterized by reduced stool frequency. We have recently correlated this dysfunction with enteric glial cell reactivity and the loss of specific neuronal populations in the enteric nervous system [1]. In the present study we investigated the mechanism of MPTP induced neurodegeneration in the enteric nervous system.

MPTP induced toxicity is dependent on the uptake of its toxic metabolite MPP+ by dopamine active transporter (DAT) expressed on dopaminergic neurons. DAT expression is highest in the ileum of the gastrointestinal tract. Consistent with this tyrosine hydroxylase (TH) immunoreactive (IR) dopaminergic neurons were significantly reduced in the ileum of MPTP-lesioned mice, but unaffected in the colon where DAT expression is lower.

Although MPTP-lesioning is not generally associated with Lewy pathology, MPTP-lesioning led to a change in localization of α-synuclein in DAT-IR neurons of the ileum. MPTP treatment did not affect α-synuclein localization in the colon.

Non-dopaminergic neurons identified by the expression of neurofilament (NFM-IR neurons) were also reduced in the myenteric plexus of the ileum, but not colon of MPTP lesioned mice.

We hypothesize that MPTP-initiated loss of non-dopaminergic cell populations may in response to MPTP induced changes in α-synuclein and/or the response of enteric glial cells to MPTP induced injury. This hypothesis is consistent with a toxic insult triggering Parkinson's disease pathology in a vulnerable cell population within the gastrointestinal tract, with secondary neurodegeneration
<figure><img src="image_125.png" /></figure>

caused by the spread of α-synuclein aggregates or neuroinflammation.

## Reference

[1] Ellett et al., Sci Rep. 2016;6:30269.

## 208. Binding prions to soils impact PrPCWD recovery but not infectivity

Alsu Kuznetsova<sup>a,b</sup>, Debbie McKenzie<sup>b</sup> and Judd M. Aiken<sup>a</sup>

<sup>a</sup>Agricultural Life and Environmental Sciences Faculty, University of Alberta, Edmonton, Canada; <sup>b</sup>Faculty of Science, University of Alberta, Edmonton, Canada

CONTACT Alsu Kuznetsova alsu@ualberta.ca

## ABSTRACT

Chronic wasting disease (CWD) is a contagious prion disease affecting cervids. Soil can act as an environmental reservoir resulting in CWD transmission. CWD prions shed from animals at the preclinical and clinical stages of disease into the environment remain infectious even after prolonged periods. In this study, we used soil minerals (quartz, illite, montmorillonite (Mte)) as well as six surface soil horizons of four different soils: horizons LFH (plant litter horizon) and Ae (illite-enriched mineral horizon) of Luvisol, horizons LFH (plant litter) and Bf (illite and iron enriched mineral horizon) of Brunisol, and Ah (humic horizon) horizons of two Chernozemic soils. Collected soil samples represent soil cover of boreal, tundra (Luvisols and Brunisols) and prairie regions (Chernozems).

Long-term incubation of prions with illite, Mte and soils results in decreased recovery of PrPCWD. Binding of prions to minerals and soils became more avid and irreversible with time; PrP signal on immunoblots continuously declined until it was no longer detectable after 30 weeks incubation in soils with loamy-clay texture and Mte minerology. This continual decline of PrPCWD immunoreactivity did not correlate with prion infectivity levels as no significant differences were found in incubation periods between mice intraperitoneally inoculated with pure 1% brain homogenate and with the CWD-BH pre-incubated with quartz or Luvisolic Ae horizon for 1 and 30 weeks. After 55 weeks incubation with Chernozem and Luvisol, bound PrPCWD was undetectable by immunoblotting but remained infectious.

We show that prions bind to soils and remain infectious after extended periods of time (at least up to 55 weeks). Our findings indicate that analysis of environmental samples for PrPCWD will be complicated as

prolonged incubation and lack of detection by western blot does not correspond to a decrease in PrPCWD infectivity.

## 209. Comparative analysis of the neuroinvasion of Chronic Wasting Disease strains (Wisc-1 and H95<sup>+</sup>) in transgenic mice expressing different cellular prion proteins

Danielle Gushue<sup>a</sup>, Camilo Duque Velásquez<sup>a</sup>, Chiye Kim<sup>a</sup>, Judd Aiken<sup>b</sup> and Debbie McKenzie<sup>a</sup>

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

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

CONTACT Danielle Gushue dgushue@ualberta.ca

## ABSTRACT

Chronic wasting disease (CWD) is a contagious prion disease of free-ranging and captive cervids from North America, Scandinavia, and Korea. The transmission cycle of CWD among cervids relies on the lymphotropic nature of CWD prions and the environmental persistence of prion infectivity shed by affected animals. Multiple prion strains circulate in CWD enzootic areas and their diversity is modulated by disease transmission between cervids expressing different PrPC molecules. Prion strains neuroinvade with different efficiency and this process is dependent on the route of exposure, the infectious dose and the structural compatibility between prion conformation and host PrPC. To evaluate if the route of exposure affects strain adaptation, we compared the neuroinvasion properties of pure Wisc-1 strain and a mixture of this one with the H95<sup>+</sup> strain. We previously demonstrated these strains have distinct biological properties, including conformational differences, brain targeting areas, and transmission host range. Two transgenic mouse lines expressing PrPC allelotypes, Q95G96 or Q95S96, were exposed either orally or intraperitoneally (IP) and the phenotypic properties produced by each strain-route-host combination were compared. Consistent with previous observations in host expressing wt PrPC, the Wisc-1 strain had higher disease penetrance and shorter incubation periods compared to the H95<sup>+</sup>. The intraperitoneal route was more efficient than the oral route, which lacked complete attack rates. The incubation period of Wisc-1 doubled compared to the intracranial (IC) route. Interestingly, the differences in incubation between the host exposed to Wisc-1 or the H95<sup>+</sup> mixture were smaller than previously reported by IC route. The Wisc-1 host-specific titre in the H95<sup>+</sup> mixture is at least two-fold lower than pure Wisc-1, suggesting the route of exposure influenced strain selection from the mixture. This also indicates Wisc-1 is