# Process of structural diversification and synergy between prion subassemblies during host adaptation

Angelique Egalon, Mohammed Moudjou, Florent Laferrière, Philippe Tixador, Jan Bohl, Mathieu Mezache, Tina Knäpple, Laetitia Herzog, Fabienne Reine, Marie Doumic, et al.

► To cite this version:

Angelique Egalon, Mohammed Moudjou, Florent Laferrière, Philippe Tixador, Jan Bohl, et al. Process of structural diversification and synergy between prion subassemblies during host adaptation. Prion 2019, Prion, 13 (S1), pp.15-16, 2019. [hal-02617741](https://hal.inrae.fr/hal-02617741)

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Prion

ISSN: 1933-6896 (Print) 1933-690X (Online) Journal homepage: www.tandfonline.com/journals/kprn20

# PRION 2019 emerging concepts

**To cite this article:** (2019) PRION 2019 emerging concepts, Prion, 13:sup1, 1-141, DOI: 10.1080/19336896.2019.1615197

**To link to this article:** https://doi.org/10.1080/19336896.2019.1615197

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© 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.

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Published online: 18 May 2019.

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Prion

ISSN: 1933-6896 (Print) 1933-690X (Online) Journal homepage: www.tandfonline.com/journals/kprn20

# PRION 2019 emerging concepts

**To cite this article:** (2019) PRION 2019 emerging concepts, Prion, 13:sup1, 1-141, DOI: 10.1080/19336896.2019.1615197

**To link to this article:** https://doi.org/10.1080/19336896.2019.1615197

<figure><img src="image_3.png" /></figure>

© 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.

<figure><img src="image_4.png" /></figure>

Published online: 18 May 2019.

<figure><img src="image_5.png" /></figure>

Submit your article to this journal

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

Article views: 14382

<figure><img src="image_8.png" /></figure>

View related articles

<figure><img src="image_9.png" /></figure>

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CrossMark

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# PRION 2019 emerging concepts

<figure><img src="image_10.png" /></figure>

OPEN ACCESS

<figure><img src="image_11.png" /></figure>

Check for updates

## Co- Editors:

David Westaway

Centre for Prions and Protein misfolding Diseases,
University of Alberta

Hermann Schaetzl

Calgary Prion Research Unit, University of Calgary

Kevin Keough

Alberta Prion Research Institute, Alberta Innovates

Dear attendees,

Welcome to Prion 2019 in downtown Edmonton.

With over 320 participants from 25 countries, we hope you are greatly stimulated by this conference. Over the coming days, 47 oral presentations, over 200 posters, two workshops and three special sessions will deliver cutting-edge concepts to understand and control debilitating prion and related neurodegenerative diseases. We hope you make the acquaintance of new theories, new technologies and - most importantly - new colleagues and collaborators.

We thank all our co-organizers and we wish you a warm welcome from the province and people of Alberta.

David, Hermann and Kevin

## 1. Interspecies transmission of the chronic wasting disease agent

Justin Greenlee

Virus and Prion Research Unit, National Animal Disease Center,
USDA Agriculture Research Service

### ABSTRACT

The presentation will summarize the results of various studies conducted at our research center that assess the transmissibility of the chronic wasting disease (CWD) agent to cattle, pigs, raccoons, goats, and sheep. This will include specifics of the relative attack rates, clinical signs, and microscopic lesions with emphasis on how to differentiate cross-species transmission of the CWD agent from the prion diseases that naturally occur in hosts such as cattle or sheep. Briefly, the relative

difficulty of transmitting the CWD agent to sheep and goats will be contrasted with the relative ease of transmitting the scrapie agent to white-tailed deer.

## 2. RT-QuIC seed amplification assays in the diagnosis of prion diseases, synucleinopathies and tauopathies

Byron Caughey

Senior Investigator at Rocky Mountain Laboratories, NIAID, NIH

### ABSTRACT

Antemortem diagnoses of many proteopathies can be difficult, due in part to an inability to detect specific causative misfolded protein aggregates with sufficient sensitivity, specificity and practicality. To address this issue we and others have developed RT-QuIC assays that exploit the ability of such aggregates to seed the polymerization of protein monomers into amyloid fibrils. PrP-based RT-QuIC assays for are available for virtually all prion diseases of mammals and have been adapted to multiple specimens including, most recently, the skin and eyes of CJD cases. Analyses of CSF and nasal brushings can give nearly 100% accurate *ante-mortem* diagnosis of sCJD. We and others have adapted the seed amplification approach to the detection of synucleinopathies such as Parkinson's disease. αSyn RT-QuIC analyses of patients' CSF can detect α-synuclein seeds early in the clinical phase of disease and has provided unprecedented diagnostic accuracy. We have also developed three types of ultrasensitive tau RT-QuIC assays optimized for (i) Alzheimer disease and chronic traumatic encephalopathy (i.e., 3R/4R tauopathies), (ii) Pick disease (a 3R tauopathy), or (iii) progressive supranuclear palsy and other 4R tauopathies. Each assay provides strong selectivity for the type of tauopathy for which it was optimized. Such selectivities may underpin the propagation of specific tau aggregate conformers *in vivo*. In summary, these assays can facilitate diagnosis and allow measurements of proteopathic biomarkers. This, in turn, may promote the therapeutic trials by improving patient cohort selection and longitudinal assessments of drug efficacy.
# PRION 2019 emerging concepts

<figure><img src="image_14.png" /></figure>

OPEN ACCESS

<figure><img src="image_15.png" /></figure>

Check for updates

**Co- Editors:**

David Westaway

Centre for Prions and Protein misfolding Diseases,
University of Alberta

Hermann Schaetzl

Calgary Prion Research Unit, University of Calgary

Kevin Keough

Alberta Prion Research Institute, Alberta Innovates

Dear attendees,

Welcome to Prion 2019 in downtown Edmonton.

With over 320 participants from 25 countries, we hope you are greatly stimulated by this conference. Over the coming days, 47 oral presentations, over 200 posters, two workshops and three special sessions will deliver cutting-edge concepts to understand and control debilitating prion and related neurodegenerative diseases. We hope you make the acquaintance of new theories, new technologies and - most importantly - new colleagues and collaborators.

We thank all our co-organizers and we wish you a warm welcome from the province nd people of Alberta.

David, Hermann and Kevin

## 1. Interspecies transmission of the chronic wasting disease agent

Justin Greenlee

Virus and Prion Research Unit, National Animal Disease Center,
USDA Agriculture Research Service

### ABSTRACT

The presentation will summarize the results of various studies conducted at our research center that assess the transmissibility of the chronic wasting disease (CWD) agent to cattle, pigs, raccoons, goats, and sheep. This will include specifics of the relative attack rates, clinical signs, and microscopic lesions with emphasis on how to differentiate cross-species transmission of the CWD agent from the prion diseases that naturally occur in hosts such as cattle or sheep. Briefly, the relative

difficulty of transmitting the CWD agent to sheep and goats will be contrasted with the relative ease of transmitting the scrapie agent to white-tailed deer.

## 2. RT-QuIC seed amplification assays in the diagnosis of prion diseases, synucleinopathies and tauopathies

Byron Caughey

Senior Investigator at Rocky Mountain Laboratories, NIAID, NIH

### ABSTRACT

Antemortem diagnoses of many proteopathies can be difficult, due in part to an inability to detect specific causative misfolded protein aggregates with sufficient sensitivity, specificity and practicality. To address this issue we and others have developed RT-QuIC assays that exploit the ability of such aggregates to seed the polymerization of protein monomers into amyloid fibrils. PrP-based RT-QuIC assays for are available for virtually all prion diseases of mammals and have been adapted to multiple specimens including, most recently, the skin and eyes of CJD cases. Analyses of CSF and nasal brushings can give nearly 100% accurate *ante-mortem* diagnosis of sCJD. We and others have adapted the seed amplification approach to the detection of synucleinopathies such as Parkinson's disease. αSyn RT-QuIC analyses of patients' CSF can detect α-synuclein seeds early in the clinical phase of disease and has provided unprecedented diagnostic accuracy. We have also developed three types of ultrasensitive tau RT-QuIC assays optimized for (i) Alzheimer disease and chronic traumatic encephalopathy (i.e., 3R/4R tauopathies), (ii) Pick disease (a 3R tauopathy), or (iii) progressive supranuclear palsy and other 4R tauopathies. Each assay provides strong selectivity for the type of tauopathy for which it was optimized. Such selectivities may underpin the propagation of specific tau aggregate conformers *in vivo*. In summary, these assays can facilitate diagnosis and allow measurements of proteopathic biomarkers. This, in turn, may promote the therapeutic trials by improving patient cohort selection and longitudinal assessments of drug efficacy.
<figure><img src="image_12.png" /></figure>

# 3. A perspective on prion strains and their significance

John Collinge

Department of Neurodegenerative Disease, Institute of Neurology, University College London (UCL)

## ABSTRACT

Mammalian prions are thought to comprise multimeric assemblies of misfolded cellular prion protein (PrP<sup>C</sup>) which propagate by recruitment of PrP monomers and subsequent fission. Although devoid of a nucleic acid genome, mammalian prions exist as multiple strains which can be serially propagated in laboratory animals and produce distinct patterns of disease. Since multiple strains can be maintained in the same inbred mouse line with identical PrP genes, strains cannot be encoded by differences in the primary structure of PrP and it is thought they represent structurally distinct seeds that are able to recruit host PrP<sup>C</sup> into their distinct polymeric forms. These strain-specific biological and structural properties can be maintained even following passage through an intermediate mammalian species with a different PrP primary structure. In addition to the fundamental biological interest in such protein-based inheritance, it is clear that understanding the prion strain phenomenon is fundamental to understanding prion disease pathogenesis and transmission properties. Critically, prion strains, rather than being a biological clone, appear to constitute a cloud of diverse molecular assemblies or quasispecies which can adapt and mutate under host and other selection pressures. This has an important bearing on therapeutic strategies which need to avoid the development of drug resistance. A proof of principle of targeting neuronal PrP<sup>C</sup>, rather than disease-associated assemblies, was established and our therapeutic approach using an anti-human PrP<sup>C</sup> monoclonal antibody in patients with CJD is underway. Prion strains may be considered as subtypes of misfolded PrP assemblies with the necessary properties and replication kinetics to evade cellular and other host defences, propagate exponentially and be able to act as an efficient pathogen, while other polymeric PrP forms are degraded or form highly stable aggregates that do not propagate and/or are relatively inert. The very diversity of prion quasispecies, the degree of which may itself vary between strains, may be fundamental to adaptation to a new host (and to particular tissues within it) to allow its survival and spread. This may also account for why some amyloids are infectious pathogens and others not and understanding the structural basis of these different pathobiological properties will be of major importance.

# 4. A unified model of prion infectivity and strain maintenance by cofactor selection

Surachai Supattapone

Departments of Biochemistry and Medicine, Dartmouth College

## ABSTRACT

The protein-only hypothesis predicts that infectious mammalian prions are composed solely of PrP<sup>Sc</sup>, a misfolded conformer of the normal prion protein, PrP<sup>C</sup>. However, to date, all wild-type protein-only PrP<sup>Sc</sup> preparations lack significant levels of prion infectivity. Using a systemic biochemical approach, our laboratory isolated and identified two different endogenous cofactor molecules, RNA and phosphatidylethanolamine, which facilitate the formation of prions with high levels of specific infectivity (>10<sup>6</sup>-fold greater than protein-only PrP<sup>Sc</sup>), leading us to propose to the alternative hypothesis that cofactor molecules are required to form wild-type infectious prions. In addition, we found that purified cofactor molecules restrict the strain properties of chemically-defined infectious prions[4], suggesting a “cofactor selection” model in which natural variation in the distribution of strain-specific cofactor molecules in different parts of the brain may be responsible for strain-dependent patterns of neurotropism. Recently, we have discovered that prions with parental strain properties and full specific infectivity can be restored from non-infectious protein-only PrP<sup>Sc</sup> *in vitro* (Burke, et al. PLoS Pathogens, in press). The restoration reaction is rapid, potent, and requires bank vole PrP<sup>C</sup> substrate, post-translational modifications, and cofactor molecules. These findings provide evidence for a unified model of prion infectivity in which the global structure of protein-only PrP<sup>Sc</sup> can accurately store latent infectious and strain information, but cofactor molecules control a reversible switch that can unmask full biological infectivity. The results are also consistent with the idea that prion strains select compatible cofactor molecules on the basis of their ability to maintain strain-specific PrP<sup>Sc</sup> conformations.

# 5. Structural insights into the mechanism of mammalian prion propagation

Witold K. Surewicz

Department of Physiology and Biophysics, Case Western Reserve University

## ABSTRACT

Despite recent progress in structural studies of ordered protein aggregates such as amyloid fibrils in general,
<figure><img src="image_16.png" /></figure>

# 3. A perspective on prion strains and their significance

John Collinge

Department of Neurodegenerative Disease, Institute of Neurology, University College London (UCL)

## ABSTRACT

Mammalian prions are thought to comprise multimeric assemblies of misfolded cellular prion protein (PrP<sup>C</sup>) which propagate by recruitment of PrP monomers and subsequent fission. Although devoid of a nucleic acid genome, mammalian prions exist as multiple strains which can be serially propagated in laboratory animals and produce distinct patterns of disease. Since multiple strains can be maintained in the same inbred mouse line with identical PrP genes, strains cannot be encoded by differences in the primary structure of PrP and it is thought they represent structurally distinct seeds that are able to recruit host PrP<sup>C</sup> into their distinct polymeric forms. These strain-specific biological and structural properties can be maintained even following passage through an intermediate mammalian species with a different PrP primary structure. In addition to the fundamental biological interest in such protein-based inheritance, it is clear that understanding the prion strain phenomenon is fundamental to understanding prion disease pathogenesis and transmission properties. Critically, prion strains, rather than being a biological clone, appear to constitute a cloud of diverse molecular assemblies or quasispecies which can adapt and mutate under host and other selection pressures. This has an important bearing on therapeutic strategies which need to avoid the development of drug resistance. A proof of principle of targeting neuronal PrP<sup>C</sup>, rather than disease-associated assemblies, was established and our therapeutic approach using an anti-human PrP<sup>C</sup> monoclonal antibody in patients with CJD is underway. Prion strains may be considered as subtypes of misfolded PrP assemblies with the necessary properties and replication kinetics to evade cellular and other host defences, propagate exponentially and be able to act as an efficient pathogen, while other polymeric PrP forms are degraded or form highly stable aggregates that do not propagate and/or are relatively inert. The very diversity of prion quasispecies, the degree of which may itself vary between strains, may be fundamental to adaptation to a new host (and to particular tissues within it) to allow its survival and spread. This may also account for why some amyloids are infectious pathogens and others not and understanding the structural basis of these different pathobiological properties will be of major importance.

# 4. A unified model of prion infectivity and strain maintenance by cofactor selection

Surachai Supattapone

Departments of Biochemistry and Medicine, Dartmouth College

## ABSTRACT

The protein-only hypothesis predicts that infectious mammalian prions are composed solely of PrP<sup>Sc</sup>, a misfolded conformer of the normal prion protein, PrP<sup>C</sup>. However, to date, all wild-type protein-only PrP<sup>Sc</sup> preparations lack significant levels of prion infectivity. Using a systemic biochemical approach, our laboratory isolated and identified two different endogenous cofactor molecules, RNA and phosphatidylethanolamine, which facilitate the formation of prions with high levels of specific infectivity (>10<sup>6</sup>-fold greater than protein-only PrP<sup>Sc</sup>), leading us to propose to the alternative hypothesis that cofactor molecules are required to form wild-type infectious prions. In addition, we found that purified cofactor molecules restrict the strain properties of chemically-defined infectious prions[4], suggesting a “cofactor selection” model in which natural variation in the distribution of strain-specific cofactor molecules in different parts of the brain may be responsible for strain-dependent patterns of neurotropism. Recently, we have discovered that prions with parental strain properties and full specific infectivity can be restored from non-infectious protein-only PrP<sup>Sc</sup> *in vitro* (Burke, et al. PLoS Pathogens, in press). The restoration reaction is rapid, potent, and requires bank vole PrP<sup>C</sup> substrate, post-translational modifications, and cofactor molecules. These findings provide evidence for a unified model of prion infectivity in which the global structure of protein-only PrP<sup>Sc</sup> can accurately store latent infectious and strain information, but cofactor molecules control a reversible switch that can unmask full biological infectivity. The results are also consistent with the idea that prion strains select compatible cofactor molecules on the basis of their ability to maintain strain-specific PrP<sup>Sc</sup> conformations.

# 5. Structural insights into the mechanism of mammalian prion propagation

Witold K. Surewicz

Department of Physiology and Biophysics, Case Western Reserve University

## ABSTRACT

Despite recent progress in structural studies of ordered protein aggregates such as amyloid fibrils in general,
<figure><img src="image_13.png" /></figure>

the structure of infectious mammalian prions is poorly understood and controversial. This presentation will discuss structural aspects of prion propagation, with a special focus on a model system of PrP23-144 amyloid that can reproduce in vitro the phenomena of strain multiplicity and transmissibility (seeding) barriers and is amenable to high-resolution structural characterization. Furthermore, we will discuss mass spectrometry-based approaches that can be used for structural characterization of brain-derived prions and the insights provided by these studies regarding the structural basis of prion infectivity.

## 6. Seed-induced Aβ deposition impairs adult neurogenesis in mouse models of Alzheimer's disease

Meyer-Luehmann

Department of Neurology, Medical Center-University of Freiburg, Germany and Faculty of Medicine, University if Freiburg, Germany

### ABSTRACT

Alzheimer's disease (AD) is characterized by severe neuronal loss as well as the accumulation of amyloid-β (Aβ) which ultimately leads to plaque formation. These Aβ deposits can be induced in young pre-depositing APP transgenic mice by intracerebral injection of Aβ-containing brain homogenate. Although a decline of neurogenic capacity in the brain of AD patients and AD mouse models has been reported, our understanding of whether this impairment is specifically altered by Aβ plaques is limited. Here, we find that induced Aβ deposition (Aβ seeding), representing early stages of plaque formation, leads to a dramatic decrease in adult neurogenesis in the hippocampus and olfactory bulb. We further report that the generation and maturation of newborn neurons is affected by the induction of Aβ deposition in both neurogenic niches. Notably, cell death and apoptosis occur in conjunction with Aβ seeding and the exposure to enriched environment and voluntary running reduces Aβ seeding via activated microglia and vivifies neurogenesis and proliferation.

## 7. Modifying prion spread through the CNS

Christina Sigurdson

Department of Pathology, University of California, San Diego

### ABSTRACT

Prion aggregates increase exponentially and rapidly transit through the central nervous system during prion disease, yet the driving factors from the host and the prion that ultimately govern spread are unclear.

Using a series of mouse models with alterations in the extracellular matrix or in the prion conformation, we investigated the determinants of prion propagation through the brain. By targeting heparan sulfate biosynthesis, we find that prion deposition sites shift and the survival time is prolonged in a strain dependent manner. These findings suggest that manipulating endogenous co-factors may be exploited to enhance prion clearance.

## 8. Drivers of neurotoxicity in prion diseases

Adriano Aguzzi

Director, Institute of Neuropathology, University of Zurich

### ABSTRACT

While the mechanics of prion replication have been subjected to intense studies and are becoming increasingly clear, little is known about what ultimately causes brain damage. It cannot just be mechanical damage from the accumulation of PrPSc, because the latter is present only in trace amounts – in contrast to systemic amyloidoses with enormous amyloid loads in e.g. spleen, liver or peripheral nerves. This suggests the existence of specific pathways of brain damage. If such pathways exist, their components may be exploitable as targets of therapeutic intervention. My lab has focused on the latter question using many different approaches. For example, we have studied the mechanism of action of neurotoxic antibodies against PrP. One such antibody (denominated POM1) was found to be a prion-mimetic, which elicits the same cellular programmes as bona fide prion infections, and can be antagonized by compounds which protect against prion toxicity. Currently, we are exploring the biogenesis of spongiform changes, arguably the most typical consequence of prion disease. It turns out that neuronal vacuolation, the basis of spongiform changes, is controlled by cellular checkpoint molecules that are subverted by prion infection and by prion-mimetic antibodies. The nature of said checkpoints will be discussed.

## 9. Targeting proteopathic seeds in Alzheimer's disease

Mathias Jucker

Department of Cellular Neurology, Hertie Institute for Clinical Brain Research, University of Tübingen, and German Center for Neurodegenerative Diseases (DZNE), D-72076 Tübingen, Germany
<figure><img src="image_17.png" /></figure>

the structure of infectious mammalian prions is poorly understood and controversial. This presentation will discuss structural aspects of prion propagation, with a special focus on a model system of PrP23-144 amyloid that can reproduce in vitro the phenomena of strain multiplicity and transmissibility (seeding) barriers and is amenable to high-resolution structural characterization. Furthermore, we will discuss mass spectrometry-based approaches that can be used for structural characterization of brain-derived prions and the insights provided by these studies regarding the structural basis of prion infectivity.

## 6. Seed-induced Aβ deposition impairs adult neurogenesis in mouse models of Alzheimer's disease

Meyer-Luehmann

Department of Neurology, Medical Center-University of Freiburg, Germany and Faculty of Medicine, University if Freiburg, Germany

### ABSTRACT

Alzheimer's disease (AD) is characterized by severe neuronal loss as well as the accumulation of amyloid-β (Aβ) which ultimately leads to plaque formation. These Aβ deposits can be induced in young pre-depositing APP transgenic mice by intracerebral injection of Aβ-containing brain homogenate. Although a decline of neurogenic capacity in the brain of AD patients and AD mouse models has been reported, our understanding of whether this impairment is specifically altered by Aβ plaques is limited. Here, we find that induced Aβ deposition (Aβ seeding), representing early stages of plaque formation, leads to a dramatic decrease in adult neurogenesis in the hippocampus and olfactory bulb. We further report that the generation and maturation of newborn neurons is affected by the induction of Aβ deposition in both neurogenic niches. Notably, cell death and apoptosis occur in conjunction with Aβ seeding and the exposure to enriched environment and voluntary running reduces Aβ seeding via activated microglia and vivifies neurogenesis and proliferation.

## 7. Modifying prion spread through the CNS

Christina Sigurdson

Department of Pathology, University of California, San Diego

### ABSTRACT

Prion aggregates increase exponentially and rapidly transit through the central nervous system during prion disease, yet the driving factors from the host and the prion that ultimately govern spread are unclear.

Using a series of mouse models with alterations in the extracellular matrix or in the prion conformation, we investigated the determinants of prion propagation through the brain. By targeting heparan sulfate biosynthesis, we find that prion deposition sites shift and the survival time is prolonged in a strain dependent manner. These findings suggest that manipulating endogenous co-factors may be exploited to enhance prion clearance.

## 8. Drivers of neurotoxicity in prion diseases

Adriano Aguzzi

Director, Institute of Neuropathology, University of Zurich

### ABSTRACT

While the mechanics of prion replication have been subjected to intense studies and are becoming increasingly clear, little is known about what ultimately causes brain damage. It cannot just be mechanical damage from the accumulation of PrPSc, because the latter is present only in trace amounts – in contrast to systemic amyloidoses with enormous amyloid loads in e.g. spleen, liver or peripheral nerves. This suggests the existence of specific pathways of brain damage. If such pathways exist, their components may be exploitable as targets of therapeutic intervention. My lab has focused on the latter question using many different approaches. For example, we have studied the mechanism of action of neurotoxic antibodies against PrP. One such antibody (denominated POM1) was found to be a prion-mimetic, which elicits the same cellular programmes as bona fide prion infections, and can be antagonized by compounds which protect against prion toxicity. Currently, we are exploring the biogenesis of spongiform changes, arguably the most typical consequence of prion disease. It turns out that neuronal vacuolation, the basis of spongiform changes, is controlled by cellular checkpoint molecules that are subverted by prion infection and by prion-mimetic antibodies. The nature of said checkpoints will be discussed.

## 9. Targeting proteopathic seeds in Alzheimer's disease

Mathias Jucker

Department of Cellular Neurology, Hertie Institute for Clinical Brain Research, University of Tübingen, and German Center for Neurodegenerative Diseases (DZNE), D-72076 Tübingen, Germany
<figure><img src="image_14.png" /></figure>

**10. Prion accumulation in the bone marrow: the origin of prionemia?**

Olivier Andreoletti<sup>a</sup>, Jean-Yves Douet<sup>b</sup>, Alvina Huor<sup>b</sup>, Séverine Lugan<sup>b</sup>, Naima Aron<sup>b</sup>, Cécile Tillier<sup>b</sup>, Hervé Cassard<sup>b</sup> and Olivier Andreoletti<sup>b</sup>

ABSTRACT

The presence of significant amount of Prion infectivity
in the bone marrow (BM) of both sCJD and vCJD
affected patients has been demonstrated. This raised
questions about the role of BM in the occurrence of
prionemia and the risk of disease transmission asso-
ciated with this tissue. To address these questions, we
developed a bone marrow transplantation model in
PrP<sup>+/+</sup> and PrP<sup>0/0</sup> C57Bl<sub>6</sub> mice. Despite the presence
of significant amount of infectivity in the bone marrow
of RML infected PrP<sup>+/+</sup> C57Bl6 mice, the reconstitu-
tion of sub-lethally γ-irradiated mice (8.5 Gy) with this
tissue failed to transmit Prion disease or to cause a
detectable prionemia. The RML peripheral inoculation
of irradiated and BM reconstituted mice clearly demon-
strated that the prion replication capacity of the radio-
sensitive BM cell populations has no detectable impact
on the occurrence and the level of Prionemia. It also
indicated that the radio resistant cells compartment is
associated with the vast majority of the infectivity in the
BM. In RML inoculated LT<sup>-/-</sup> / TNFα<sup>-/-</sup> C57Bl<sub>6</sub>, prion
accumulated in Bone Marrow tissue but mice displayed
a very low/undetectable prionemia. Together these
results support the contention that conversely to BM,
germinal lymphoid centers play a central role in the
occurrence and the level of prionemia.

11. Arguments for Alzheimer's and Parkinson's
diseases caused by prions

Stanley B. Prusiner

Institute of Neurodegenerative Diseases, and Professor of Neurology
and Biochemistry, University of California San Francisco

ABSTRACT

Arguments for Alzheimer's (AD) and Parkinson's dis-
eases (PD) being caused by prions continue to advance
with new evidence. Findings in the brains of deceased
AD patients argue that both Aβ and tau prions can be
demonstrated by bioassays in cultured cells as well as in
transgenic (Tg) mice. Likewise, studies of the brains of

deceased MSA patients have been found to contain α-synuclein prions by bioassays in cultured cells and Tg mice. Conversely, the brains of AD patients do not contain α-synuclein prions, and the brains of MSA patients do not contain Aβ or tau prions. Additionally, while the brains of patients who died of either progressive supranuclear palsy (PSP) or cortico-basal degeneration (CBD) contained tau prions, neither Aβ nor α-synuclein prions were detectable. Merely measuring the levels of Aβ, tau, and α-synuclein appears to give misleading information about the etiology and pathogenesis of neurodegenerative diseases (NDs). From a large array of bioassays, we conclude that AD, PD, MSA, and the frontotemporal dementias, including PSP and CBD, are all prion diseases. Our findings argue that changes in the conformations of Aβ, tau, and α-synuclein underlie the acquisition of prion infectivity in all of these NDs.

12. Genetic risk factors for sporadic CJD:
replication, expression, function

Simon Mead

UCL Institute of Prion Diseases and NIHR Senior Investigator

ABSTRACT

By definition, sporadic Creutzfeldt-Jakob disease is not
caused by genetic mutation of the prion protein gene,
and the patient has no identifiable environmental expo-
sure. Like common neurodegenerative dementias, age is
a powerful risk factor, but the molecular mechanisms of
increasing risk with advanced age are unclear. We
research both genomic and epigenomic factors in
human prion disease by genome-wide association
study. Last year we completed initial analysis of genome
wide association study with contributions of patient
samples from UK, USA, Germany, France, Italy,
Netherlands, Austria, Spain, Switzerland and
Australia. We have now analysed over 5,000 case sam-
ples and a large number of controls. Additional to
PRNP, we identified other genetic loci at which multi-
ple SNPs surpass statistical thresholds for genome-wide
significance. A SNP at one of these loci, near to STX6,
is already known to be a genetic risk factor for a
tauopathy, progressive supranuclear palsy. We have
followed up these findings by replication, investigation
of correlations with clinical phenotypes, allelic expres-
sion of genes in blood and brain and in experimental
models. Genetic risk factors point to critical aspects of
the pathobiology of prion disease and should be
assessed for their potential as therapeutic targets.

<footnote><sup>a</sup>INRA Research Director, UMR INRA ENVT 1225; <sup>b</sup>UMR INRA ENVT 1225, Interactions Hôtes Agents Pathogènes, 23 chemin des capelles 31076 Toulouse, FRANCE</footnote>
<figure><img src="image_18.png" /></figure>

**10. Prion accumulation in the bone marrow: the origin of prionemia?**

Olivier Andreoletti<sup>a</sup>, Jean-Yves Douet<sup>b</sup>, Alvina Huor<sup>b</sup>, Séverine Lugan<sup>b</sup>, Naima Aron<sup>b</sup>, Cécile Tillier<sup>b</sup>, Hervé Cassard<sup>b</sup> and Olivier Andreoletti<sup>b</sup>

**ABSTRACT**

The presence of significant amount of Prion infectivity in the bone marrow (BM) of both sCJD and vCJD affected patients has been demonstrated. This raised questions about the role of BM in the occurrence of prionemia and the risk of disease transmission associated with this tissue. To address these questions, we developed a bone marrow transplantation model in PrP<sup>+/+</sup> and PrP<sup>0/0</sup> C57Bl<sub>6</sub> mice. Despite the presence of significant amount of infectivity in the bone marrow of RML infected PrP<sup>+/+</sup> C57Bl6 mice, the reconstitution of sub-lethally γ-irradiated mice (8.5 Gy) with this tissue failed to transmit Prion disease or to cause a detectable prionemia. The RML peripheral inoculation of irradiated and BM reconstituted mice clearly demonstrated that the prion replication capacity of the radiosensitive BM cell populations has no detectable impact on the occurrence and the level of Prionemia. It also indicated that the radio resistant cells compartment is associated with the vast majority of the infectivity in the BM. In RML inoculated LT<sup>-/-</sup> / TNFα<sup>-/-</sup> C57Bl<sub>6</sub>, prion accumulated in Bone Marrow tissue but mice displayed a very low/undetectable prionemia. Together these results support the contention that conversely to BM, germinal lymphoid centers play a central role in the occurrence and the level of prionemia.

**11. Arguments for Alzheimer's and Parkinson's diseases caused by prions**

Stanley B. Prusiner

Institute of Neurodegenerative Diseases, and Professor of Neurology and Biochemistry, University of California San Francisco

**ABSTRACT**

Arguments for Alzheimer's (AD) and Parkinson's diseases (PD) being caused by prions continue to advance with new evidence. Findings in the brains of deceased AD patients argue that both Aβ and tau prions can be demonstrated by bioassays in cultured cells as well as in transgenic (Tg) mice. Likewise, studies of the brains of

deceased MSA patients have been found to contain α-synuclein prions by bioassays in cultured cells and Tg mice. Conversely, the brains of AD patients do not contain α-synuclein prions, and the brains of MSA patients do not contain Aβ or tau prions. Additionally, while the brains of patients who died of either progressive supranuclear palsy (PSP) or corticobasal degeneration (CBD) contained tau prions, neither Aβ nor α-synuclein prions were detectable. Merely measuring the levels of Aβ, tau, and α-synuclein appears to give misleading information about the etiology and pathogenesis of neurodegenerative diseases (NDs). From a large array of bioassays, we conclude that AD, PD, MSA, and the frontotemporal dementias, including PSP and CBD, are all prion diseases. Our findings argue that changes in the conformations of Aβ, tau, and α-synuclein underlie the acquisition of prion infectivity in all of these NDs.

**12. Genetic risk factors for sporadic CJD: replication, expression, function**

Simon Mead

UCL Institute of Prion Diseases and NIHR Senior Investigator

**ABSTRACT**

By definition, sporadic Creutzfeldt-Jakob disease is not caused by genetic mutation of the prion protein gene, and the patient has no identifiable environmental exposure. Like common neurodegenerative dementias, age is a powerful risk factor, but the molecular mechanisms of increasing risk with advanced age are unclear. We research both genomic and epigenomic factors in human prion disease by genome-wide association study. Last year we completed initial analysis of genome wide association study with contributions of patient samples from UK, USA, Germany, France, Italy, Netherlands, Austria, Spain, Switzerland and Australia. We have now analysed over 5,000 case samples and a large number of controls. Additional to PRNP, we identified other genetic loci at which multiple SNPs surpass statistical thresholds for genome-wide significance. A SNP at one of these loci, near to STX6, is already known to be a genetic risk factor for a tauopathy, progressive supranuclear palsy. We have followed up these findings by replication, investigation of correlations with clinical phenotypes, allelic expression of genes in blood and brain and in experimental models. Genetic risk factors point to critical aspects of the pathobiology of prion disease and should be assessed for their potential as therapeutic targets.

<footnote><sup>a</sup>INRA Research Director, UMR INRA ENVT 1225; <sup>b</sup>UMR INRA ENVT 1225, Interactions Hôtes Agents Pathogènes, 23 chemin des capelles 31076 Toulouse, FRANCE</footnote>
<figure><img src="image_15.png" /></figure>

# 13. A solid-state of conceptualization of information transfer from gene to message protein

Steve McKnight

Department of Biochemistry UT Southwestern

## ABSTRACT

My presentation will describe speculative ideas and early stage research concerning the flow of genetic information from the nuclear residence of genes to the disparate, cytoplasmic sites of protein synthesis. This process is particularly important for neurons, in which the nuclear location of genes may be far-removed from localized sites of mRNA translation at active synapses. I propose that this process of information transfer is meticulously guided by transient structures formed from protein segments of low sequence complexity or intrinsic disorder. These low complexity domains are ubiquitously associated with regulatory proteins that control gene expression and RNA biogenesis, but they are also found in the central channel of nuclear pores, the nexus points of intermediate filament assembly, and the locations of action of other well-studied cellular proteins and pathways. Upon being organized into localized cellular positions via mechanisms utilizing properly folded protein domains, thereby facilitating elevated local concentration, certain low complexity domains adopt cross-β interactions as described 60-70 years ago by Linus Pauling. Derivation of the atomic structure of labile polymers formed from the low complexity domain of the fused in sarcoma (FUS) RNA binding protein has demonstrated how these cross-β interactions can be both structurally specific and labile to disassembly. These weakly tethered assemblies, I propose, are built to relay the passage of genetic information from one site to another within a cell, ensuring that the process is of extreme fidelity.

# 14. Beyond neurofibrillary tangles in tauopathy

Karen Hsiao Ashe and N. Bud Grossman

Center for Memory Research and Care, Edmund Wallace and Anne Marie Tulloch Chairs in Neurology and Neuroscience at the University of Minnesota

## ABSTRACT

Soluble forms of tau, unrelated to neurofibrillary tangles, play pathogenic roles in tauopathies. A soluble tau fragment, Δtau314 that resists forming fibrils forms when caspase-2 (CASP2) cleaves human tau at Asp314. Δtau314 is elevated in forebrain tissue of

humans with mild cognitive impairment, Alzheimer's disease, Huntington's disease and Lewy body dementia. This cleavage event, along with tau phosphorylation, leads to tau mislocalization and reduced AMPA receptor levels in dendritic spines and abnormal post-synaptic function (reduced mEPSCs). Rendering endogenous mouse tau (mTau) non-cleavable by CASP2 by introducing a homologous mutation in mTau, Asp303-to-Glu reduces mTau in dendritic spines, increases excitatory synaptic transmission and resistance to excitotoxin-induced seizures, and ameliorates premature mortality in transgenic mice expressing human amyloid precursor protein. These data support a role for non-amyloid forms of proteins present in the neuropathological amyloid-containing structures (e.g., tangles) that characterize their respective diseases, and suggest a strategy for repairing synaptic transmission in tauopathies by inhibiting CASP2.

# 15. Cellular prion infection: from traffic jams to new drug targets

Sabine Gilch

Canada Research Chair in Prion Disease Research, University of Calgary

## ABSTRACT

Prion infection on a cellular level is characterized by the presence of $PrP^{Sc}$ aggregates at the plasma membrane, in vesicles along the endo-lysosomal pathway and in recycling endosomes, as well as an increased level of unesterified cholesterol. High cholesterol content can affect membrane properties and vesicle trafficking. Our goal is to understand the impact of prion infection on endo-lysosomal vesicle trafficking, to define a link with aberrant cholesterol metabolism and to validate new therapeutic compounds that target those pathways. We show that prion infection induces a 'traffic jam' by impairing with retrograde cargo transport and lysosomal degradation capacity, processes that require functions of the retromer complex and rab7, respectively. We show that stimulating retrograde transport, lysosomal degradation capacity and enhancing cholesterol efflux in neuronal cells reduces prion propagation. Overall, our studies provide new insights into prion-induced cellular dysfunction. We observed alterations similar to those described in Alzheimer's disease, indicating that the endo-lysosomal system might be an intersection in the pathogenesis of those neurodegenerative diseases.
<figure><img src="image_19.png" /></figure>

# 13. A solid-state of conceptualization of information transfer from gene to message protein

Steve McKnight

Department of Biochemistry UT Southwestern

## ABSTRACT

My presentation will describe speculative ideas and early stage research concerning the flow of genetic information from the nuclear residence of genes to the disparate, cytoplasmic sites of protein synthesis. This process is particularly important for neurons, in which the nuclear location of genes may be far-removed from localized sites of mRNA translation at active synapses. I propose that this process of information transfer is meticulously guided by transient structures formed from protein segments of low sequence complexity or intrinsic disorder. These low complexity domains are ubiquitously associated with regulatory proteins that control gene expression and RNA biogenesis, but they are also found in the central channel of nuclear pores, the nexus points of intermediate filament assembly, and the locations of action of other well-studied cellular proteins and pathways. Upon being organized into localized cellular positions via mechanisms utilizing properly folded protein domains, thereby facilitating elevated local concentration, certain low complexity domains adopt cross-β interactions as described 60-70 years ago by Linus Pauling. Derivation of the atomic structure of labile polymers formed from the low complexity domain of the fused in sarcoma (FUS) RNA binding protein has demonstrated how these cross-β interactions can be both structurally specific and labile to disassembly. These weakly tethered assemblies, I propose, are built to relay the passage of genetic information from one site to another within a cell, ensuring that the process is of extreme fidelity.

# 14. Beyond neurofibrillary tangles in tauopathy

Karen Hsiao Ashe and N. Bud Grossman

Center for Memory Research and Care, Edmund Wallace and Anne Marie Tulloch Chairs in Neurology and Neuroscience at the University of Minnesota

## ABSTRACT

Soluble forms of tau, unrelated to neurofibrillary tangles, play pathogenic roles in tauopathies. A soluble tau fragment, Δtau314 that resists forming fibrils forms when caspase-2 (CASP2) cleaves human tau at Asp314. Δtau314 is elevated in forebrain tissue of

humans with mild cognitive impairment, Alzheimer's disease, Huntington's disease and Lewy body dementia. This cleavage event, along with tau phosphorylation, leads to tau mislocalization and reduced AMPA receptor levels in dendritic spines and abnormal post-synaptic function (reduced mEPSCs). Rendering endogenous mouse tau (mTau) non-cleavable by CASP2 by introducing a homologous mutation in mTau, Asp303-to-Glu reduces mTau in dendritic spines, increases excitatory synaptic transmission and resistance to excitotoxin-induced seizures, and ameliorates premature mortality in transgenic mice expressing human amyloid precursor protein. These data support a role for non-amyloid forms of proteins present in the neuropathological amyloid-containing structures (e.g., tangles) that characterize their respective diseases, and suggest a strategy for repairing synaptic transmission in tauopathies by inhibiting CASP2.

# 15. Cellular prion infection: from traffic jams to new drug targets

Sabine Gilch

Canada Research Chair in Prion Disease Research, University of Calgary

## ABSTRACT

Prion infection on a cellular level is characterized by the presence of $PrP^{Sc}$ aggregates at the plasma membrane, in vesicles along the endo-lysosomal pathway and in recycling endosomes, as well as an increased level of unesterified cholesterol. High cholesterol content can affect membrane properties and vesicle trafficking. Our goal is to understand the impact of prion infection on endo-lysosomal vesicle trafficking, to define a link with aberrant cholesterol metabolism and to validate new therapeutic compounds that target those pathways. We show that prion infection induces a 'traffic jam' by impairing with retrograde cargo transport and lysosomal degradation capacity, processes that require functions of the retromer complex and rab7, respectively. We show that stimulating retrograde transport, lysosomal degradation capacity and enhancing cholesterol efflux in neuronal cells reduces prion propagation. Overall, our studies provide new insights into prion-induced cellular dysfunction. We observed alterations similar to those described in Alzheimer's disease, indicating that the endo-lysosomal system might be an intersection in the pathogenesis of those neurodegenerative diseases.
<figure><img src="image_16.png" /></figure>

# 16. Design and optimization of nucleic acid derivatives for inhibition of prion-like propagation at W32 site of SOD1 enzyme in Amyotrophic Lateral Sclerosis

Vijaya Kumar Hinge<sup>a,b</sup>, Nikolay Blinov<sup>a,b</sup>, Dipankar Roy<sup>a,b</sup>, W. Ted Allison<sup>a</sup>, David S. Wishart<sup>a</sup> and Andriy Kovalenko<sup>a,b</sup>

CONTACT Vijaya Kumar Hinge hinge@ualberta.ca

## ABSTRACT

Misfolding of human Cu/Zn superoxide dismutase (SOD1) cytosolic metalloenzyme results in the formation of neurotoxic oligomers in familial Amyotrophic Lateral Sclerosis (fALS). Over 180 mutations have been identified and linked to the gene encoding of SOD1 in fALS. Currently, there is no cure for ALS and only two Food and Drug Administration (FDA)-approved drugs, Riluzole and Edaravone, are available for treatment of ALS patients. The human cell culture studies demonstrated that misfolded SOD1 propagates from cell to cell in a prion-like fashion and W32 residue is important for templated misfolding of wild type SOD1 (wtSOD1) into toxic aggregates [1]. The recent animal studies on ALS Zebrafish embryo model suggest that mutation of W32S decreases the toxicity to motor neuron functions [2]. The inhibition of pathological misfolding of SOD1 via drug-like molecules binding at W32 site is a novel and promising therapeutic strategy for the development of ALS therapy.

In *silico* de novo drug design and pharmacophore-restrained high-throughput virtual screening (HTVS) methods were used to identify the anti-ALS lead candidates at W32 site. A library of commercially available lead-like compounds (650,000) and Food and Drug Administration (FDA) approved small molecules were screened at W32 site of SOD1 using HTVS and were filtered through a uracil-based pharmacophore model. Based on the analysis of physico-chemical descriptors and pharmacophore features of the experimental binding mode of 5-FUrd at W32, a group of new Anti-ALS lead compounds were rationally designed. Anti-ALS lead candidates were ranked with in-house quantitative structure-activity relationship (QSAR) models based on the 3D-RISM-KH molecular solvation theory derived molecular descriptors which accurately predicted the blood brain barrier (BBB) permeability [3]. A new protocol was developed to account for structural solvation effects and was successfully validated via predicting experimental binding modes of 5-FUrd at W32 site of

SOD1. The protocol combines the water placement algorithm based on the 3D-RISM-KH molecular theory of solvation and molecular docking simulations implemented in the Molecular Operating Environment (MOE) integrated drug discovery package. One of the anti-ALS lead compound, Telbivudine, tested in ALS-Zebrafish embryo model significantly rescued axonopathy in a dose-dependent manner [2,4]. The newly designed lead-like compounds were selected by virtual screening, and *de novo* drug design methods show higher affinity compared to the Telbivudine and can potentially inhibit prion-like propagation of misfolding of SOD1.

## References

[1] Grad L, Guest WC, Yanai A, et al. Intermolecular transmission of superoxide dismutase 1 misfolding in living cells. Proc Natl Acad Sci USA. 2011;108:16,398-403.

[2] Duval et al. Neurobiology of disease 2019;124:297-310.

[3] Roy et al. ACS Omega. 2019 (accepted).

[4] Treatment of amyotrophic lateral sclerosis (ALS). Patent application no 62/637,013. USPTO (pending); 2018.

# 17. Large-scale production of phospholipid cofactor recombinant prions with high specific infectivity

Daniel J. Walsh<sup>a</sup>, Marcus D. Tuttle<sup>b</sup>, Cassandra M. Burke<sup>a</sup>, Kurt W. Zilm<sup>b</sup>, and Surachai Supattapone<sup>a,d</sup>

CONTACT Daniel J. Walsh daniel.j.walsh.GR@dartmouth.edu

## ABSTRACT

The fundamental event underlying mammalian prion infectivity is the induced conformational change of PrP<sup>C</sup> into PrP<sup>Sc</sup>. A major goal in the field is to determine the structural mechanism of this conversion process, and a critical step towards this goal would be to determine the protein structure of fully infectious PrP<sup>Sc</sup>. One technique that could be used to determine the structure of infectious PrP<sup>Sc</sup> with high resolution is solid-state (ss)NMR, but application of this method requires the production of milligram quantities of a homogeneous, isotopically-labelled PrP<sup>Sc</sup> sample whose specific infectivity is equivalent to that of authentic brain-derived PrP27-30 (~10<sup>6</sup> LD<sub>50</sub>/µg PrP) [1].

We previously used sPMCA to produce two chemically defined recombinant (rec)PrP<sup>Sc</sup> conformers originally

<footnote><sup>a</sup>University of Alberta, Edmonton, Canada; <sup>b</sup>Nanotechnology Research Centre, Edmonton, Canada</footnote>

<footnote><sup>a</sup>Departments of Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA; <sup>b</sup>Department of Chemistry, Yale University, New Haven, Connecticut, USA; <sup>c</sup>Medicine, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA</footnote>
<figure><img src="image_20.png" /></figure>

# 16. Design and optimization of nucleic acid derivatives for inhibition of prion-like propagation at W32 site of SOD1 enzyme in Amyotrophic Lateral Sclerosis

Vijaya Kumar Hinge<sup>a,b</sup>, Nikolay Blinov<sup>a,b</sup>, Dipankar Roy<sup>a,b</sup>, W. Ted Allison<sup>a</sup>, David S. Wishart<sup>a</sup> and Andriy Kovalenko<sup>a,b</sup>

CONTACT Vijaya Kumar Hinge hinge@ualberta.ca

## ABSTRACT

Misfolding of human Cu/Zn superoxide dismutase (SOD1) cytosolic metalloenzyme results in the formation of neurotoxic oligomers in familial Amyotrophic Lateral Sclerosis (fALS). Over 180 mutations have been identified and linked to the gene encoding of SOD1 in fALS. Currently, there is no cure for ALS and only two Food and Drug Administration (FDA)-approved drugs, Riluzole and Edaravone, are available for treatment of ALS patients. The human cell culture studies demonstrated that misfolded SOD1 propagates from cell to cell in a prion-like fashion and W32 residue is important for templated misfolding of wild type SOD1 (wtSOD1) into toxic aggregates [1]. The recent animal studies on ALS Zebrafish embryo model suggest that mutation of W32S decreases the toxicity to motor neuron functions [2]. The inhibition of pathological misfolding of SOD1 via drug-like molecules binding at W32 site is a novel and promising therapeutic strategy for the development of ALS therapy.

In *silico* de novo drug design and pharmacophore-restrained high-throughput virtual screening (HTVS) methods were used to identify the anti-ALS lead candidates at W32 site. A library of commercially available lead-like compounds (650,000) and Food and Drug Administration (FDA) approved small molecules were screened at W32 site of SOD1 using HTVS and were filtered through a uracil-based pharmacophore model. Based on the analysis of physico-chemical descriptors and pharmacophore features of the experimental binding mode of 5-FUrd at W32, a group of new Anti-ALS lead compounds were rationally designed. Anti-ALS lead candidates were ranked with in-house quantitative structure-activity relationship (QSAR) models based on the 3D-RISM-KH molecular solvation theory derived molecular descriptors which accurately predicted the blood brain barrier (BBB) permeability [3]. A new protocol was developed to account for structural solvation effects and was successfully validated via predicting experimental binding modes of 5-FUrd at W32 site of

SOD1. The protocol combines the water placement algorithm based on the 3D-RISM-KH molecular theory of solvation and molecular docking simulations implemented in the Molecular Operating Environment (MOE) integrated drug discovery package. One of the anti-ALS lead compound, Telbivudine, tested in ALS-Zebrafish embryo model significantly rescued axonopathy in a dose-dependent manner [2,4]. The newly designed lead-like compounds were selected by virtual screening, and *de novo* drug design methods show higher affinity compared to the Telbivudine and can potentially inhibit prion-like propagation of misfolding of SOD1.

## References

[1] Grad L, Guest WC, Yanai A, et al. Intermolecular transmission of superoxide dismutase 1 misfolding in living cells. Proc Natl Acad Sci USA. 2011;108:16,398-403.

[2] Duval et al. Neurobiology of disease 2019;124:297-310.

[3] Roy et al. ACS Omega. 2019 (accepted).

[4] Treatment of amyotrophic lateral sclerosis (ALS). Patent application no 62/637,013. USPTO (pending); 2018.

# 17. Large-scale production of phospholipid cofactor recombinant prions with high specific infectivity

Daniel J. Walsh<sup>a</sup>, Marcus D. Tuttle<sup>b</sup>, Cassandra M. Burke<sup>a</sup>, Kurt W. Zilm<sup>b</sup>, and Surachai Supattapone<sup>a,d</sup>

CONTACT Daniel J. Walsh daniel.j.walsh.GR@dartmouth.edu

## ABSTRACT

The fundamental event underlying mammalian prion infectivity is the induced conformational change of PrP<sup>C</sup> into PrP<sup>Sc</sup>. A major goal in the field is to determine the structural mechanism of this conversion process, and a critical step towards this goal would be to determine the protein structure of fully infectious PrP<sup>Sc</sup>. One technique that could be used to determine the structure of infectious PrP<sup>Sc</sup> with high resolution is solid-state (ss)NMR, but application of this method requires the production of milligram quantities of a homogeneous, isotopically-labelled PrP<sup>Sc</sup> sample whose specific infectivity is equivalent to that of authentic brain-derived PrP27-30 (~10<sup>6</sup> LD<sub>50</sub>/µg PrP) [1].

We previously used sPMCA to produce two chemically defined recombinant (rec)PrP<sup>Sc</sup> conformers originally

<footnote><sup>a</sup>University of Alberta, Edmonton, Canada; <sup>b</sup>Nanotechnology Research Centre, Edmonton, Canada</footnote>

<footnote><sup>a</sup>Departments of Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA; <sup>b</sup>Department of Chemistry, Yale University, New Haven, Connecticut, USA; <sup>c</sup>Medicine, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA</footnote>
<figure><img src="image_17.png" /></figure>

derived from the same seed. Whereas cofactor recPrP<sup>Sc</sup> produced with phospholipids displayed high levels of specific infectivity, protein-only recPrP<sup>Sc</sup> produced from PrP alone was completely non-infectious [2]. A limitation of PMCA technology is that it can only be used to produce microgram quantities of PrP<sup>Sc</sup>, and therefore is not a feasible option for generating ssNMR samples. To overcome this limitation, we produced large quantities of cofactor recPrP<sup>Sc</sup> and protein-only recPrP<sup>Sc</sup> by shaking rather than PMCA. Shaking-produced cofactor recPrP<sup>Sc</sup> caused scrapie in both wild-type mice and bank voles (homozygous M109), while protein-only recPrP<sup>Sc</sup> failed to infect either species. End-point titration bioassays indicated that the specific infectivity of shaking-produced cofactor recPrP<sup>Sc</sup> was ~10<sup>6</sup> LD<sub>50</sub>/µg PrP, while that of protein-only recPrP<sup>Sc</sup> was <5 LD<sub>50</sub>/µg PrP. These results confirm that shaking-produced phospholipid cofactor recPrP<sup>Sc</sup> is a good candidate for structural studies of bona fide mammalian prion infectivity. The morphologies of cofactor recPrP<sup>Sc</sup> and protein-only recPrP<sup>Sc</sup> molecules could not be easily distinguished by negative-stain electron microscopy, but differences in the secondary structure between the two conformers can be detected by ssNMR.

## References

[1] Deleault NR, Harris BT, Rees JR, et al. Formation of native prions from minimal componenets in vitro. *Proc Natl Acad Sci U S A.* 2007;104(23):9741–9746.

[2] Deleault NR, Walsh DJ, Piro JR, et al. Cofactor molecules maintain infectious conformation and restrict strain properties in purified prions. *Proc Natl Acad Sci U S A.* 2012;109(28):E1938–E1946.

## 18. Targeting protein aggregation through nanoparticle-based inhibitors

Bibin G. Anand<sup>a,b,c</sup>, Kailash Prasad Prajapathi<sup>b</sup>, Kriti Dubey<sup>b</sup> and karunakar Kar<sup>b</sup>

Abstract not available.

## 19. Amplification of prions derived from single cells isolated from different brain regions of RML infected animals by laser capture microdissection

D. Gorski<sup>a</sup>, A. Lyon<sup>a</sup>, A. Mukherjee<sup>a</sup>, S. Medina<sup>b</sup>, S. Booth<sup>b</sup>, S. Pritzkow<sup>a</sup> and C. Soto<sup>a</sup>

and Special Pathogens, National Microbiology Laboratory, Public Health Agency of Canada

CONTACT D. Gorski Damian.A.Gorski@uth.tmc.edu

## ABSTRACT

Prion diseases are uniformly fatal neurodegenerative disorders characterized by the misfolding and aggregation of host-encoded prion protein (PrP<sup>C</sup>) into a β-sheet rich, insoluble, and protease-resistant conformation termed PrP<sup>Sc</sup>, which serves as the sole infectious agent. Depending on the prion disease, PrP<sup>Sc</sup> can adopt different isoforms, which preferentially deposit in distinct brain regions and differentially accumulate in the neurons, astroglia, and microglia of these areas. Currently, the cellular factors and processes governing the differential cellular vulnerability to PrP<sup>Sc</sup> deposition and damage amongst different cell types and brain regions are not known. The putative differences in biochemical, biological, and structural characteristics of PrP<sup>Sc</sup> associated with different cells and regions is also yet to be described. Indeed, several previous reports have shown that different prion strains can co-exist in the same brain and may be masked by a single dominant pathological phenotype. In this study, we optimized the protein misfolding cyclic amplification (PMCA) technology to amplify PrP<sup>Sc</sup> from a single cell. For these studies we used a cell line chronically infected with RML prions and dilute it to add individual cells per sample. Serial dilutions of infected cell culture showed that PMCA enabled amplification of prions from a single cell. This was further confirmed by plating cell dilutions. The presence of a single cell was confirmed via microscopy. We then applied this technique to amplify PrP<sup>Sc</sup> from individual cells extracted from the brain of prion infected animals via laser capture dissection. We show successful amplification of prions from individual neurons from some areas of the brain and are currently analysing the presence of PrPSc in neurons and glial cells extracted via LCM from the cerebellum, thalamus, cortex, and hippocampus of RML infected mice. Partial fixation and staining of the tissue prior to LCM did not interfere with PMCA amplification. This technology will enable to study the characteristics of prions associated with different cell types isolated from distinct areas of the brain of animals and humans infected with different prion strains. Experiments can be done comparing PrP<sup>Sc</sup> in neuronal versus glial cell types originating from distinct brain regions as well distinct time points of disease progression. These findings can then be coupled with single cell genomics and proteomics analysis. These findings will shed light on the molecular basis of differential cellular vulnerability to prion infection and the nerve tropism of distinct prion strains.

<footnote><sup>a</sup>Department of Bioscience and Bioengineering, Indian Institute of Technology Jodhpur, India; <sup>b</sup>School of Life science Jawaharlal Nehru University, New Delhi, India; <sup>c</sup>Center for Prions and Protein folding diseases, University of Alberta, Edmonton, Canada</footnote>

<footnote><sup>a</sup>Mitchell Center for Alzheimer's disease and related Brain disorders, University of Texas Medical School at Houston; <sup>b</sup>Zoonotic Diseases</footnote>
<figure><img src="image_21.png" /></figure>

derived from the same seed. Whereas cofactor $recPrP^{Sc}$ produced with phospholipids displayed high levels of specific infectivity, protein-only $recPrP^{Sc}$ produced from PrP alone was completely non-infectious [2]. A limitation of PMCA technology is that it can only be used to produce microgram quantities of $PrP^{Sc}$, and therefore is not a feasible option for generating ssNMR samples. To overcome this limitation, we produced large quantities of cofactor $recPrP^{Sc}$ and protein-only $recPrP^{Sc}$ by shaking rather than PMCA. Shaking-produced cofactor $recPrP^{Sc}$ caused scrapie in both wild-type mice and bank voles (homozygous M109), while protein-only $recPrP^{Sc}$ failed to infect either species. End-point titration bioassays indicated that the specific infectivity of shaking-produced cofactor $recPrP^{Sc}$ was ~$10^6$ LD₅₀/µg PrP, while that of protein-only $recPrP^{Sc}$ was <5 LD₅₀/µg PrP. These results confirm that shaking-produced phospholipid cofactor $recPrP^{Sc}$ is a good candidate for structural studies of bona fide mammalian prion infectivity. The morphologies of cofactor $recPrP^{Sc}$ and protein-only $recPrP^{Sc}$ molecules could not be easily distinguished by negative-stain electron microscopy, but differences in the secondary structure between the two conformers can be detected by ssNMR.

## References

[1] Deleault NR, Harris BT, Rees JR, et al. Formation of native prions from minimal componenets in vitro. *Proc Natl Acad Sci U S A.* 2007;104(23):9741-9746.

[2] Deleault NR, Walsh DJ, Piro JR, et al. Cofactor molecules maintain infectious conformation and restrict strain properties in purified prions. *Proc Natl Acad Sci U S A.* 2012;109(28):E1938-E1946.

## 18. Targeting protein aggregation through nanoparticle-based inhibitors

Bibin G. Anand<sup>a,b,c</sup>, Kailash Prasad Prajapathi<sup>b</sup>, Kriti Dubey<sup>b</sup> and karunakar Kar<sup>b</sup>

Abstract not available.

## 19. Amplification of prions derived from single cells isolated from different brain regions of RML infected animals by laser capture microdissection

D. Gorski<sup>a</sup>, A. Lyon<sup>a</sup>, A. Mukherjee<sup>a</sup>, S. Medina<sup>b</sup>, S. Booth<sup>b</sup>, S. Pritzkow<sup>a</sup> and C. Soto<sup>a</sup>

and Special Pathogens, National Microbiology Laboratory, Public Health Agency of Canada

CONTACT D. Gorski Damian.A.Gorski@uth.tmc.edu

## ABSTRACT

Prion diseases are uniformly fatal neurodegenerative disorders characterized by the misfolding and aggregation of host-encoded prion protein ($PrP^C$) into a β-sheet rich, insoluble, and protease-resistant conformation termed $PrP^{Sc}$, which serves as the sole infectious agent. Depending on the prion disease, $PrP^{Sc}$ can adopt different isoforms, which preferentially deposit in distinct brain regions and differentially accumulate in the neurons, astroglia, and microglia of these areas. Currently, the cellular factors and processes governing the differential cellular vulnerability to $PrP^{Sc}$ deposition and damage amongst different cell types and brain regions are not known. The putative differences in biochemical, biological, and structural characteristics of $PrP^{Sc}$ associated with different cells and regions is also yet to be described. Indeed, several previous reports have shown that different prion strains can co-exist in the same brain and may be masked by a single dominant pathological phenotype. In this study, we optimized the protein misfolding cyclic amplification (PMCA) technology to amplify $PrP^{Sc}$ from a single cell. For these studies we used a cell line chronically infected with RML prions and dilute it to add individual cells per sample. Serial dilutions of infected cell culture showed that PMCA enabled amplification of prions from a single cell. This was further confirmed by plating cell dilutions. The presence of a single cell was confirmed via microscopy. We then applied this technique to amplify $PrP^{Sc}$ from individual cells extracted from the brain of prion infected animals via laser capture dissection. We show successful amplification of prions from individual neurons from some areas of the brain and are currently analysing the presence of $PrPSc$ in neurons and glial cells extracted via LCM from the cerebellum, thalamus, cortex, and hippocampus of RML infected mice. Partial fixation and staining of the tissue prior to LCM did not interfere with PMCA amplification. This technology will enable to study the characteristics of prions associated with different cell types isolated from distinct areas of the brain of animals and humans infected with different prion strains. Experiments can be done comparing $PrP^{Sc}$ in neuronal versus glial cell types originating from distinct brain regions as well distinct time points of disease progression. These findings can then be coupled with single cell genomics and proteomics analysis. These findings will shed light on the molecular basis of differential cellular vulnerability to prion infection and the nerve tropism of distinct prion strains.

<footnote><sup>a</sup>Department of Bioscience and Bioengineering, Indian Institute of Technology Jodhpur, India; <sup>b</sup>School of Life science Jawaharlal Nehru University, New Delhi, India; <sup>c</sup>Center for Prions and Protein folding diseases, University of Alberta, Edmonton, Canada</footnote>

<footnote><sup>a</sup>Mitchell Center for Alzheimer's disease and related Brain disorders, University of Texas Medical School at Houston; <sup>b</sup>Zoonotic Diseases</footnote>
<figure><img src="image_18.png" /></figure>

**20. New insights into the structure of bovine spongiform encephalopathy (BSE) prions**

Razieh Kamali-Jamil<sup>a,b</sup>, Sara Amidian<sup>a,b</sup>, Brian Tancowny<sup>a,b</sup>, Xiongyao Wang<sup>a,b</sup>, Ester Vázquez-Fernández<sup>a,b</sup>, Xinli Tang<sup>a</sup>, Howard Young<sup>b</sup> and Holger Wille<sup>a,b</sup>

CONTACT Razieh Kamali-Jamil kamalija@ualberta.ca

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

**ABSTRACT**

**Background:** Bovine spongiform encephalopathy (BSE) is caused by the conversion of the cellular prion protein (PrP<sup>C</sup>) to the infectious form (PrP<sup>Sc</sup>). Prion diseases in cattle include classical BSE (C-type BSE), and atypical strains known as H-type and C-type BSE. Consumption of BSE-infected meat products may have caused variant Creutzfeldt-Jakob disease in humans. Previously, the structure of the recombinant prion protein has been analysed using X-ray crystallography and NMR spectroscopy methods. However, the PrP<sup>Sc</sup> has hampered all attempts at structural determination due to its insolubility and propensity to aggregate. A recent cryo-EM study on glycosylphosphatidylinositol-anchorless PrP<sup>Sc</sup> proposed a four-rung β-solenoid architecture for individual PrP<sup>Sc</sup> molecules. Currently, no three-dimensional structure is available for BSE prions. The aim of this project is the characterization and detailed analysis of the structure of BSE prions.

**Materials and Methods:** L-type BSE prions were purified from the brains of prion-infected transgenic mice expressing bovine PrP<sup>C</sup>, using phosphotungstate anions followed by sucrose gradient centrifugation. The purification quality was assessed using SDS PAGE followed by silver staining or Western blotting. The purified samples were analysed via negative stain electron microscopy to determine the quality of the sample preparations. We also developed an immunogold labeling protocol for the specific detection of BSE fibrils on electron microscopy grids. Next, electron micrographs of individual BSE fibrils were analysed through a variety of image processing techniques in order to obtain three-dimensional reconstructions. For this purpose, fibril images with a clear helical twist were selected, segmented, aligned, classified, and oriented. Subsequently, a three-dimensional volume could be generated through back projection.

**Results and Conclusion:** The successful purification of BSE prion fibrils was confirmed by Western blotting and negative stain EM. Image analyses revealed a heterogeneous population of BSE fibrils consisting of two intertwined protofibrils, in agreement with the results of previous studies. Additionally, fibrils with only one protofilament were observed, which is a novel finding. 2D class averages on more than 500 fibrils further confirmed the two distinct morphologies of the L-type BSE fibrils. We postulate that the two-protofilament BSE fibrils result from the interaction of two individual fibrils. Thus, our findings reveal differences on the quaternary structure level. Lastly, these observations will be helpful for future efforts to obtain high-resolution information on the structure of BSE prions.

**21. Proximity-dependent biotinylation identifies protein interactors of pathological tau and synuclein in human ESC-derived neurons**

Tagan A. Griffin, Elisa M. Cleveland, Robert W. Newberry, George A. Carlson, Paul D. Schnier and Stanley B. Prusiner

Institute for Neurodegenerative Diseases, Weill Institute for Neurosciences, University of California, San Francisco, CA, USA

CONTACT Tagan A. Griffin tagan.griffin@ucsf.edu

**ABSTRACT**

Tau and synuclein can act as prions, undergoing templated conformational changes resulting in the accumulation of aggregated proteins, which drives the spread of neurodegeneration throughout the brain. It is unclear which other cellular proteins contribute to this process. To investigate this question, we utilized a promiscuous biotin ligase (BioID2) fused to tau or synuclein to biotinylate interacting proteins in human embryonic stem cell (ESC)-derived neurons following treatment with disease-causing recombinant fibrils (tau K18 or synuclein preformed fibrils [PFFs]). To identify interactors in a relevant cell type, we first generated a stable human ESC line (H1 ESCs) expressing neurogenin 2 (NGN2) under the control of an inducible promoter using PiggyBac transposon vectors. Upon doxycycline induction, this ESC line differentiated into a nearly pure population of glutamatergic neurons in under 3 weeks. Additional vectors were used to generate ESC lines that differentiated into neurons expressing tau-BioID2 (WT or P301L), synuclein-BioID2 (WT or A53T), and a control line expressing BioID2 alone. Following neuronal differentiation, recombinant fibrils were added to the culture media to induce aggregation, and cells were harvested 24 h or 3 weeks later. Following

<footnote><sup>a</sup>Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Canada; <sup>b</sup>Department of Biochemistry, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, Canada</footnote>
<figure><img src="image_22.png" /></figure>

**20. New insights into the structure of bovine spongiform encephalopathy (BSE) prions**

Razieh Kamali-Jamil<sup>a,b</sup>, Sara Amidian<sup>a,b</sup>, Brian Tancowny<sup>a,b</sup>, Xiongyao Wang<sup>a,b</sup>, Ester Vázquez-Fernández<sup>a,b</sup>, Xinli Tang<sup>a</sup>, Howard Young<sup>b</sup> and Holger Wille<sup>a,b</sup>

CONTACT Razieh Kamali-Jamil kamalija@ualberta.ca

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

**ABSTRACT**

**Background:** Bovine spongiform encephalopathy (BSE) is caused by the conversion of the cellular prion protein (PrP<sup>C</sup>) to the infectious form (PrP<sup>Sc</sup>). Prion diseases in cattle include classical BSE (C-type BSE), and atypical strains known as H-type and C-type BSE. Consumption of BSE-infected meat products may have caused variant Creutzfeldt-Jakob disease in humans. Previously, the structure of the recombinant prion protein has been analysed using X-ray crystallography and NMR spectroscopy methods. However, the PrP<sup>Sc</sup> has hampered all attempts at structural determination due to its insolubility and propensity to aggregate. A recent cryo-EM study on glycosylphosphatidylinositol-anchorless PrP<sup>Sc</sup> proposed a four-rung β-solenoid architecture for individual PrP<sup>Sc</sup> molecules. Currently, no three-dimensional structure is available for BSE prions. The aim of this project is the characterization and detailed analysis of the structure of BSE prions.

**Materials and Methods:** L-type BSE prions were purified from the brains of prion-infected transgenic mice expressing bovine PrP<sup>C</sup>, using phosphotungstate anions followed by sucrose gradient centrifugation. The purification quality was assessed using SDS PAGE followed by silver staining or Western blotting. The purified samples were analysed via negative stain electron microscopy to determine the quality of the sample preparations. We also developed an immunogold labeling protocol for the specific detection of BSE fibrils on electron microscopy grids. Next, electron micrographs of individual BSE fibrils were analysed through a variety of image processing techniques in order to obtain three-dimensional reconstructions. For this purpose, fibril images with a clear helical twist were selected, segmented, aligned, classified, and oriented. Subsequently, a three-dimensional volume could be generated through back projection.

**Results and Conclusion:** The successful purification of BSE prion fibrils was confirmed by Western blotting and negative stain EM. Image analyses revealed a heterogeneous population of BSE fibrils consisting of two intertwined protofibrils, in agreement with the results of previous studies. Additionally, fibrils with only one protofilament were observed, which is a novel finding. 2D class averages on more than 500 fibrils further confirmed the two distinct morphologies of the L-type BSE fibrils. We postulate that the two-protofilament BSE fibrils result from the interaction of two individual fibrils. Thus, our findings reveal differences on the quaternary structure level. Lastly, these observations will be helpful for future efforts to obtain high-resolution information on the structure of BSE prions.

**21. Proximity-dependent biotinylation identifies protein interactors of pathological tau and synuclein in human ESC-derived neurons**

Tagan A. Griffin, Elisa M. Cleveland, Robert W. Newberry, George A. Carlson, Paul D. Schnier and Stanley B. Prusiner

Institute for Neurodegenerative Diseases, Weill Institute for Neurosciences, University of California, San Francisco, CA, USA

CONTACT Tagan A. Griffin tagan.griffin@ucsf.edu

**ABSTRACT**

Tau and synuclein can act as prions, undergoing templated conformational changes resulting in the accumulation of aggregated proteins, which drives the spread of neurodegeneration throughout the brain. It is unclear which other cellular proteins contribute to this process. To investigate this question, we utilized a promiscuous biotin ligase (BioID2) fused to tau or synuclein to biotinylate interacting proteins in human embryonic stem cell (ESC)-derived neurons following treatment with disease-causing recombinant fibrils (tau K18 or synuclein preformed fibrils [PFFs]). To identify interactors in a relevant cell type, we first generated a stable human ESC line (H1 ESCs) expressing neurogenin 2 (NGN2) under the control of an inducible promoter using PiggyBac transposon vectors. Upon doxycycline induction, this ESC line differentiated into a nearly pure population of glutamatergic neurons in under 3 weeks. Additional vectors were used to generate ESC lines that differentiated into neurons expressing tau-BioID2 (WT or P301L), synuclein-BioID2 (WT or A53T), and a control line expressing BioID2 alone. Following neuronal differentiation, recombinant fibrils were added to the culture media to induce aggregation, and cells were harvested 24 h or 3 weeks later. Following

<footnote><sup>a</sup>Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Canada; <sup>b</sup>Department of Biochemistry, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, Canada</footnote>
<figure><img src="image_19.png" /></figure>

denaturing lysis and streptavidin purification, the biotinylated proteins were subjected to quantitative mass spectrometry using tandem-mass tag (TMT 10-plex) labels, enabling direct comparisons between multiple conditions (WT vs. mutant protein, ± fibrils, and early vs. late time points). We identified a number of known tau and synuclein interactors as well as previously unreported candidate interactors, many of which were less abundantly biotinylated in neurons treated with fibrils relative to neurons not treated with fibrils. This was accompanied by a striking increase in the abundance of biotinylated tau or synuclein, consistent with aggregate formation and a concomitant loss of physiological interactions. We also found several other biotinylated proteins whose abundances increase following the addition of fibrils. Tau-BioID2 and synuclein-BioID2 shared few interactors in common, consistent with a model of prion replication driven by tau or synuclein alone. These results identify novel candidate interactors of tau and synuclein, and shed light on the mechanisms of prion infection and replication in human neurons.

**22. Incapability of mutant PrP<sup>Sc</sup> to recruit wild-type PrP<sup>C</sup> in a familial prion disease linked to PrP<sup>Q227X</sup> mutation implies a role of GPI anchor in prion propagation**

Pingping Shen<sup>a,b</sup>, Johnny Dang<sup>a</sup>, Zerui Wang<sup>a,b</sup>, Jue Yuan<sup>a</sup>, Yue Lang<sup>a,b</sup>, Jiachun Feng<sup>b</sup>, Qingzhong Kong<sup>a,b,c</sup>, Annemiek J. M. Rozemuller<sup>d</sup>, Li Cui<sup>b</sup>, Robert B. Petersen<sup>a,b</sup>, Wen-Quan Zou<sup>a,b,e</sup>

**CONTACT** Wen-Quan Zou wxz6@case.edu

**ABSTRACT**

Cellular prion protein (PrP<sup>C</sup>) is a glycoprotein anchored to the cell surface by a glycosylphosphatidylinositol (GPI) anchor. The key molecular event in the pathogenesis of prion disease is the conversion of PrP<sup>C</sup> into its pathological isoform PrP<sup>Sc</sup>. Several lines of evidence have indicated that this event takes place on the cell surface and alternation of PrP<sup>C</sup> localization on the cell surface by mediating the GPI anchor dramatically affects the formation and infectivity of PrP<sup>Sc</sup>. Notably, anchorless PrP in transgenic (Tg) mice produced deposition of PrP amyloid in the brain, but caused no neurological signs in animals. A patient with an anchorless PrP<sup>Q227X</sup> stop codon mutation was reported to

exhibit a GSS-like prion disease phenotype. Here we examined the anchorless PrP from the brain of the patient with PrP<sup>Q227X</sup> mutation and compared the physicochemical properties of secreted and un-secreted PrP isoforms from a transfected human neuron line expressing PrP<sup>Q227X</sup>. Epitope mapping revealed for the first time that the PrP<sup>Sc</sup> from the patient with Q227X was derived from the mutant allele only. Fractionation of cultured human neuroblastoma cells expressing human wild-type or Q227X mutant PrP demonstrated most wild-type PrP (PrP<sup>WT</sup>) being recovered in the cell lysate fraction, and most mutant PrP<sup>Q227X</sup> recovered in the medium fraction, suggesting that PrP<sup>WT</sup> was attached to the cell surface, while PrP<sup>Q227X</sup> was secreted into the media. In addition, the mutant PrP lacked the N-linked glycans found in PrP<sup>WT</sup>. Two small fragments migrating at approximately ~6–8 kDa were identified in the culture media, but not in cell lysate fraction. Finally, typical PrP<sup>Sc</sup> three bands were amplified with normal humanized transgenic mouse brain as the PrP<sup>C</sup> substrate by serial protein misfolding cyclic amplification. Our finding that PrP<sup>Sc</sup> derived from the mutant GPI-anchorless PrP failed to recruit cell surface-attached PrP<sup>WT</sup> suggests that the GPI anchor plays an important role in prion propagation when mutant and wild-type alleles are present *in vivo*.

**Funding**

Supported in part by the CJD Foundation and the National Institutes of Health (NIH) NS062787 and NS087588 to W. Q.Z., NS062787 and NS109532 to W.Q.Z., and Q.K., NS088604 to Q.K. as well as the National Natural Science Foundation of China [No. 81,671,186] to LC.

**23. Inferring the effects of deer population and landscape structure on spread of the chronic wasting disease in Alberta**

Peter Smolko<sup>a</sup>, Dana Seidel<sup>b</sup>, Evelyn Merrill<sup>a</sup>, Margo Pybus<sup>a,c</sup>, Anne Hubbs<sup>c</sup> and Mark Ball<sup>c</sup>

**CONTACT** Peter Smolko smolko@ualberta.ca

**ABSTRACT**

Chronic wasting disease (CWD) is a 100% fatal prion disease of cervids that has been detected in wild deer and elk and moose in two Canadian provinces. Alberta has had a surveillance programme for CWD since 1996. CWD in wild deer in Canada was detected in adjacent Saskatchewan (2000) and later in Alberta (2005). Based

<footnote><sup>a</sup>Department of Pathology and Neurology, Case Western Reserve University, Cleveland, OH, USA; <sup>b</sup>Department of Neurology, First Hospital of Jilin University, Changchun, Jilin Province, China; <sup>c</sup>National Prion Disease Pathology Surveillance Center, Case Western Reserve University, Cleveland, OH, USA; <sup>d</sup>Dutch Surveillance Center for Prion Diseases, University Medical Center Utrecht Heidelberglaan 100, Utrecht, and Netherlands Brain Bank, Amsterdam, The Netherlands; <sup>e</sup>Foundation of Science, Central Michigan University College of Medicine, Mount Pleasant, MI, USA</footnote>

<footnote><sup>a</sup>Department of Biological Sciences, University of Alberta, Edmonton, AB, Canada; <sup>b</sup>Department of Environmental Science, Policy & Management, University of California Berkeley, CA, USA; <sup>c</sup>Alberta Fish & Wildlife Division, Government of Alberta, Edmonton, AB, Canada</footnote>
<figure><img src="image_23.png" /></figure>

denaturing lysis and streptavidin purification, the biotinylated proteins were subjected to quantitative mass spectrometry using tandem-mass tag (TMT 10-plex) labels, enabling direct comparisons between multiple conditions (WT vs. mutant protein, ± fibrils, and early vs. late time points). We identified a number of known tau and synuclein interactors as well as previously unreported candidate interactors, many of which were less abundantly biotinylated in neurons treated with fibrils relative to neurons not treated with fibrils. This was accompanied by a striking increase in the abundance of biotinylated tau or synuclein, consistent with aggregate formation and a concomitant loss of physiological interactions. We also found several other biotinylated proteins whose abundances increase following the addition of fibrils. Tau-BioID2 and synuclein-BioID2 shared few interactors in common, consistent with a model of prion replication driven by tau or synuclein alone. These results identify novel candidate interactors of tau and synuclein, and shed light on the mechanisms of prion infection and replication in human neurons.

**22. Incapability of mutant $PrP^{Sc}$ to recruit wild-type $PrP^C$ in a familial prion disease linked to $PrP^{Q227X}$ mutation implies a role of GPI anchor in prion propagation**

Pingping Shen<sup>a,b</sup>, Johnny Dang<sup>a</sup>, Zerui Wang<sup>a,b</sup>, Jue Yuan<sup>a</sup>, Yue Lang<sup>a,b</sup>, Jiachun Feng<sup>b</sup>, Qingzhong Kong<sup>a,b,c</sup>, Annemiek J. M. Rozemuller<sup>d</sup>, Li Cui<sup>b</sup>, Robert B. Petersen<sup>a,b</sup>, Wen-Quan Zou<sup>a,b,e</sup>

**CONTACT** Wen-Quan Zou wxz6@case.edu

**ABSTRACT**

Cellular prion protein ($PrP^C$) is a glycoprotein anchored to the cell surface by a glycosylphosphatidylinositol (GPI) anchor. The key molecular event in the pathogenesis of prion disease is the conversion of $PrP^C$ into its pathological isoform $PrP^{Sc}$. Several lines of evidence have indicated that this event takes place on the cell surface and alternation of $PrP^C$ localization on the cell surface by mediating the GPI anchor dramatically affects the formation and infectivity of $PrP^{Sc}$. Notably, anchorless PrP in transgenic (Tg) mice produced deposition of PrP amyloid in the brain, but caused no neurological signs in animals. A patient with an anchorless $PrP^{Q227X}$ stop codon mutation was reported to

exhibit a GSS-like prion disease phenotype. Here we examined the anchorless PrP from the brain of the patient with $PrP^{Q227X}$ mutation and compared the physicochemical properties of secreted and un-secreted PrP isoforms from a transfected human neuron line expressing $PrP^{Q227X}$. Epitope mapping revealed for the first time that the $PrP^{Sc}$ from the patient with Q227X was derived from the mutant allele only. Fractionation of cultured human neuroblastoma cells expressing human wild-type or Q227X mutant PrP demonstrated most wild-type PrP ($PrP^{WT}$) being recovered in the cell lysate fraction, and most mutant $PrP^{Q227X}$ recovered in the medium fraction, suggesting that $PrP^{WT}$ was attached to the cell surface, while $PrP^{Q227X}$ was secreted into the media. In addition, the mutant PrP lacked the N-linked glycans found in $PrP^{WT}$. Two small fragments migrating at approximately ~6–8 kDa were identified in the culture media, but not in cell lysate fraction. Finally, typical $PrP^{Sc}$ three bands were amplified with normal humanized transgenic mouse brain as the $PrP^C$ substrate by serial protein misfolding cyclic amplification. Our finding that $PrP^{Sc}$ derived from the mutant GPI-anchorless PrP failed to recruit cell surface-attached $PrP^{WT}$ suggests that the GPI anchor plays an important role in prion propagation when mutant and wild-type alleles are present in vivo.

**Funding**

Supported in part by the CJD Foundation and the National Institutes of Health (NIH) NS062787 and NS087588 to W. Q.Z., NS062787 and NS109532 to W.Q.Z., and Q.K., NS088604 to Q.K. as well as the National Natural Science Foundation of China [No. 81,671,186] to LC.

**23. Inferring the effects of deer population and landscape structure on spread of the chronic wasting disease in Alberta**

Peter Smolko<sup>a</sup>, Dana Seidel<sup>b</sup>, Evelyn Merrill<sup>a</sup>, Margo Pybus<sup>a,c</sup>, Anne Hubbs<sup>c</sup> and Mark Ball<sup>c</sup>

**CONTACT** Peter Smolko smolko@ualberta.ca

**ABSTRACT**

Chronic wasting disease (CWD) is a 100% fatal prion disease of cervids that has been detected in wild deer and elk and moose in two Canadian provinces. Alberta has had a surveillance programme for CWD since 1996. CWD in wild deer in Canada was detected in adjacent Saskatchewan (2000) and later in Alberta (2005). Based

<footnote><sup>a</sup>Department of Pathology and Neurology, Case Western Reserve University, Cleveland, OH, USA; <sup>b</sup>Department of Neurology, First Hospital of Jilin University, Changchun, Jilin Province, China; <sup>c</sup>National Prion Disease Pathology Surveillance Center, Case Western Reserve University, Cleveland, OH, USA; <sup>d</sup>Dutch Surveillance Center for Prion Diseases, University Medical Center Utrecht Heidelberglaan 100, Utrecht, and Netherlands Brain Bank, Amsterdam, The Netherlands; <sup>e</sup>Foundation of Science, Central Michigan University College of Medicine, Mount Pleasant, MI, USA</footnote>

<footnote><sup>a</sup>Department of Biological Sciences, University of Alberta, Edmonton, AB, Canada; <sup>b</sup>Department of Environmental Science, Policy & Management, University of California Berkeley, CA, USA; <sup>c</sup>Alberta Fish & Wildlife Division, Government of Alberta, Edmonton, AB, Canada</footnote>
<figure><img src="image_20.png" /></figure>

on data collected during the Alberta surveillance programme we assessed the risk of a harvested mule deer and white-tailed deer having CWD over a 15-year period (2000–2016) in eastern Alberta. We hypothesized that the probability of a hunter harvested deer being CWD positive was a function of time since the disease was first detected in the area (2000), sex and age of the deer and characteristics of the area where the deer was harvested (agricultural areas, extent of woody cover, distance to rivers and streams, topography, road density or distance to human settlements, and proximity to other positive CWD cases). We used previously developed risk model using surveillance data from 2000–2012 (n = 162 CWD cases) to predict risk in deer harvested in 2013–2016. We then redeveloped the risk model using data from 2000–2016 (n = 591) and tested the predictions of this model using positives from 2017 (n = 327). We also assessed how species, sex, importance of site characteristics, and distance to known CWD cases changed over time in influencing predicted risk of a deer having CWD in the core area and on the edge of the CWD surveillance zone. The top ranked model supported a pattern of increasing prevalence of CWD in hunter harvested deer over time. The risk among mule deer was higher compared to white-tailed deer and among males compared to females; however, the difference between species decreased and between sexes increased compared to early period. The CWD risk was greater in areas close to rivers with abundant woody cover where surveillance and control efforts should be focused.

**KEYWORDS:** Mule deer; white-tailed deer; transmissible spongiform encephalopathy; prion-protein; prevalence; disease management

## 24. Effect of the exosomal prion protein on the amyloid-β aggregation kinetics

Mohsin Shafiq<sup>a*</sup>, Alexander Hartmann<sup>a*</sup>, Stefano Da Vela<sup>b*</sup>, Christiane Muth<sup>a</sup>, Behnam Mohammadi<sup>a</sup>, Luise Linsenmeier<sup>a</sup>, Hermann Altmeppen<sup>a</sup>, Susanne Krasemann<sup>a</sup>, Dimitri Svergun<sup>b</sup> and Markus Glatzel<sup>a</sup>

**CONTACT** Mohsin Shafiq m.shafiq@uke.de

*These authors contributed equally to this work.

### ABSTRACT

The cellular prion protein (PrP<sup>C</sup>), a neuronally enriched membrane protein, is shown to impart a dual role in the pathological progression of Alzheimer's disease. PrP<sup>C</sup>

molecules present on the cell surface serve as receptors for Aβ oligomers (Aβo) and initiate neurotoxic signalling events; in contrast, extracellularly shed and cleaved forms of PrP<sup>C</sup> along with exosomally expressed PrP<sup>C</sup> are suggested to accelerate the Aβ<sub>42</sub> fibril formation by sequestering the Aβo thereby playing a neuroprotective role.

Aim of the current study is to investigate the influence of PrP<sup>C</sup> expressing (WT) and PrP<sup>C</sup> deficient (KO) exosomes on the Aβ<sub>42</sub> oligomerization kinetics, utilizing small angle X-ray scattering (SAXS).

Experiments were designed for measuring the SAXS scattering profiles of various structural intermediates of Aβ<sub>42</sub> along the different time points in aggregation cascade, in the presence of PrP<sup>C</sup>-WT and PrP<sup>C</sup>-KO exosomes.

Preliminary modelling of our data shows that the presence of both exosome variants (PrP<sup>C</sup>-KO and WT) leads to accelerated Aβ<sub>42</sub> fibrillation. However, relatively higher levels of unbound in-solution Aβo were noticed in the presence of PrP-KO exosomes, indicating a decline in Aβ<sub>42</sub> binding ability of PrP<sup>C</sup>-KO exosomes compared to that of PrP<sup>C</sup>-WT exosomes. We ascribe from our data that the presence of the PrP<sup>C</sup> is crucial for the Aβ-sequestering activity in exosomes.

## 25. Aqueous extraction of formalin-fixed paraffin-embedded tissue and detection of prion disease using real-time quaking-induced conversion

Eric M. Nicholson, Trudy Tatum and Soyoun Hwang

United States Department of Agriculture, Agricultural Research Service, National Animal Disease Center, Ames, Iowa, USA

**CONTACT** Eric M. Nicholson eric.nicholson@ars.usda.gov

### ABSTRACT

Formalin-fixed paraffin-embedded tissue (FFPET) is mainstay in the diagnosis of prion disease by immuno-histochemistry (IHC), the standard by which all other TSE diagnostic protocols are judged. IHC offers advantages over diagnostic approaches that typically utilize fresh or frozen tissue with regard to sample preservation. However, IHC and the formalin-fixed tissues utilized for IHC do not typically couple well to amplification based approaches of prion disease detection. This limitation has the potential to hamper sensitivity of techniques compatible with FFPET as advances in amplification based techniques such as real-time quaking induced conversion (RT-QuIC) continue to enhance sensitivity. In this study, we apply an approach based on previous work utilizing FFPET and an aqueous extraction step for western blot and ELISA based detection of prion disease. FFPET from prion disease transmission studies of scrapie

<footnote><sup>a</sup>Institute of Neuropathology, University Medical Center Hamburg-Eppendorf (UKE), Hamburg, Germany; <sup>b</sup>EMBL European Molecular Biology Laboratory, Hamburg, Germany</footnote>
<figure><img src="image_24.png" /></figure>

on data collected during the Alberta surveillance programme we assessed the risk of a harvested mule deer and white-tailed deer having CWD over a 15-year period (2000–2016) in eastern Alberta. We hypothesized that the probability of a hunter harvested deer being CWD positive was a function of time since the disease was first detected in the area (2000), sex and age of the deer and characteristics of the area where the deer was harvested (agricultural areas, extent of woody cover, distance to rivers and streams, topography, road density or distance to human settlements, and proximity to other positive CWD cases). We used previously developed risk model using surveillance data from 2000–2012 (n = 162 CWD cases) to predict risk in deer harvested in 2013–2016. We then redeveloped the risk model using data from 2000–2016 (n = 591) and tested the predictions of this model using positives from 2017 (n = 327). We also assessed how species, sex, importance of site characteristics, and distance to known CWD cases changed over time in influencing predicted risk of a deer having CWD in the core area and on the edge of the CWD surveillance zone. The top ranked model supported a pattern of increasing prevalence of CWD in hunter harvested deer over time. The risk among mule deer was higher compared to white-tailed deer and among males compared to females; however, the difference between species decreased and between sexes increased compared to early period. The CWD risk was greater in areas close to rivers with abundant woody cover where surveillance and control efforts should be focused.

**KEYWORDS:** Mule deer; white-tailed deer; transmissible spongiform encephalopathy; prion-protein; prevalence; disease management

## 24. Effect of the exosomal prion protein on the amyloid-β aggregation kinetics

Mohsin Shafiq<sup>a*</sup>, Alexander Hartmann<sup>a*</sup>, Stefano Da Vela<sup>b*</sup>, Christiane Muth<sup>a</sup>, Behnam Mohammadi<sup>a</sup>, Luise Linsenmeier<sup>a</sup>, Hermann Altmeppen<sup>a</sup>, Susanne Krasemann<sup>a</sup>, Dimitri Svergun<sup>b</sup> and Markus Glatzel<sup>a</sup>

**CONTACT** Mohsin Shafiq m.shafiq@uke.de

*These authors contributed equally to this work.

### ABSTRACT

The cellular prion protein (PrP<sup>C</sup>), a neuronally enriched membrane protein, is shown to impart a dual role in the pathological progression of Alzheimer's disease. PrP<sup>C</sup>

molecules present on the cell surface serve as receptors for Aβ oligomers (Aβo) and initiate neurotoxic signalling events; in contrast, extracellularly shed and cleaved forms of PrP<sup>C</sup> along with exosomally expressed PrP<sup>C</sup> are suggested to accelerate the Aβ<sub>42</sub> fibril formation by sequestering the Aβo thereby playing a neuroprotective role.

Aim of the current study is to investigate the influence of PrP<sup>C</sup> expressing (WT) and PrP<sup>C</sup> deficient (KO) exosomes on the Aβ<sub>42</sub> oligomerization kinetics, utilizing small angle X-ray scattering (SAXS).

Experiments were designed for measuring the SAXS scattering profiles of various structural intermediates of Aβ<sub>42</sub> along the different time points in aggregation cascade, in the presence of PrP<sup>C</sup>-WT and PrP<sup>C</sup>-KO exosomes.

Preliminary modelling of our data shows that the presence of both exosome variants (PrP<sup>C</sup>-KO and WT) leads to accelerated Aβ<sub>42</sub> fibrillation. However, relatively higher levels of unbound in-solution Aβo were noticed in the presence of PrP-KO exosomes, indicating a decline in Aβ<sub>42</sub> binding ability of PrP<sup>C</sup>-KO exosomes compared to that of PrP<sup>C</sup>-WT exosomes. We ascribe from our data that the presence of the PrP<sup>C</sup> is crucial for the Aβ-sequestering activity in exosomes.

## 25. Aqueous extraction of formalin-fixed paraffin-embedded tissue and detection of prion disease using real-time quaking-induced conversion

Eric M. Nicholson, Trudy Tatum and Soyoun Hwang

United States Department of Agriculture, Agricultural Research Service, National Animal Disease Center, Ames, Iowa, USA

**CONTACT** Eric M. Nicholson eric.nicholson@ars.usda.gov

### ABSTRACT

Formalin-fixed paraffin-embedded tissue (FFPET) is mainstay in the diagnosis of prion disease by immuno-histochemistry (IHC), the standard by which all other TSE diagnostic protocols are judged. IHC offers advantages over diagnostic approaches that typically utilize fresh or frozen tissue with regard to sample preservation. However, IHC and the formalin-fixed tissues utilized for IHC do not typically couple well to amplification based approaches of prion disease detection. This limitation has the potential to hamper sensitivity of techniques compatible with FFPET as advances in amplification based techniques such as real-time quaking induced conversion (RT-QuIC) continue to enhance sensitivity. In this study, we apply an approach based on previous work utilizing FFPET and an aqueous extraction step for western blot and ELISA based detection of prion disease. FFPET from prion disease transmission studies of scrapie

<footnote><sup>a</sup>Institute of Neuropathology, University Medical Center Hamburg-Eppendorf (UKE), Hamburg, Germany; <sup>b</sup>EMBL European Molecular Biology Laboratory, Hamburg, Germany</footnote>
<figure><img src="image_21.png" /></figure>

in sheep, chronic wasting disease in white-tailed deer or transmissible mink encephalopathy in cattle were cut at 5 µm thickness. Samples containing the tissue equivalent of as little as one 5 µm section can be used to readily discriminate positive from negative samples using RT-QuIC. The detection approach is avoiding the potential of a mixed chemical/biological waste stream that would occur if organic solvents were used in the removal of paraffin from the same, and offers the diagnostic and research community an addition in the diagnosis of TSEs.

**26. Exploring the structure of mouse prion fibrils by site-directed spin-labelling and ESR spectroscopy**

Kuan Yu Chu<sup>a,b</sup>, Chien Lun Hung<sup>c</sup>, Yun-Wei Chiang<sup>d</sup>, Sunney I. Chan<sup>a,e</sup> and Rita P.-Y. Chen<sup>b,d</sup>

<sup>a</sup>Department of Chemistry, College of Science, Nation Taiwan University, Taipei, Taiwan; <sup>b</sup>Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan; <sup>c</sup>Department of Chemistry, Nation Tsing Hua University, Taipei, Taiwan; <sup>d</sup>Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan; <sup>e</sup>Institute of Chemistry, Academia Sinica, Taipei, Taiwan

CONTACT Kuan Yu Chu brettchu007@gmail.com

**ABSTRACT**

**Introduction:** Recombinant prion protein (PrP) is a 23 kDa soluble protein without GPI anchor and N-glycosylation. The structure of the N-terminal domain is disordered and the structure of the C-terminal domain contains three α-helices, two β-sheets and one disulphide bond connecting helix 2 and 3. To study the structural conversion mechanism of full-length mouse prion protein (mPrP), we explore which segments of the protein are involved in this conformational transition thus form the cross-β structure.

**Methods:** Structure conversion of mPrP was studied using Electron Spin Resonance Spectroscopy (ESR) and Site-Directed Spin Labelling (SDSL). Two intrinsic Cys of mPrP were mutated to Ala. At the selected positions, the residue(s) were replaced by Cys and labelled with MTSSL. The spin-labelled PrPs were seeded by WT PrP fibrils to form various spin-labelled fibrils. The spin-diluted fibrils were prepared by mixing the spin-labelled PrP with WT PrP.

**Results:** ESR results of N174R1/N181R1, N181R1/Q186R1, and T193R1 fibrils showed that the whole helix 2 was converted into several β-strands and acted as part of the amyloid core by forming cross-β structure. For helix 3, not the whole helix 3 is transformed into cross-β structure during fibril formation. The local region around residue 212 might maintain its helicity and connect two intermolecular β-sheets. The 217–224

region formed a β-strand and are involved in the cross-β structure

**Conclusions:** Part of helix 1 of mPrP was unfolded during fibril formation. However, helix 2 and part of helix 3 of mPrP were involved in conformational conversion and formed the cross-β structure. The N-terminal part of helix 3 remained its helical structure.

**27. Biodegradation of BSE prions in compost**

Tim A. McAllister<sup>a</sup>, Shanwei Xu<sup>b</sup>, Sujeema Abeysekara<sup>a,c</sup>, Sandor Dudas<sup>c</sup>, Gordon Mitchell<sup>d</sup> and Stefanie Czub<sup>c</sup>

<sup>a</sup>Agriculture and Agri-Food Canada, Lethbridge Research and Development Centre, Lethbridge, Alberta, Canada; <sup>b</sup>Alberta Agriculture and Forestry, Lethbridge, Alberta, Canada; <sup>c</sup>Canadian and OIE Reference Laboratories for BSE, Canadian Food Inspection Agency Lethbridge Laboratory, Lethbridge, Alberta, Canada; <sup>d</sup>National and OIE Reference Laboratory for Scrapie and CWD, Canadian Food Inspection Agency, Ottawa, Ontario, Canada

CONTACT Tim A. McAllister tim.mcallister@canada.ca

**ABSTRACT**

To reduce the transmission of PrP<sup>BSE</sup>, specified risk material (SRM) that potentially contain PrP<sup>BSE</sup> is required to be removed from the food and feed chains and disposed of via rendering and landfilling or incineration in North America. However, composting could be a more cost effective means of disposing of SRM and be more suitable in areas where SRM pickup services or suitable landfills are unavailable. Our study investigated the degradation of PrP<sup>BSE</sup> (classical BSE) in lab-scale composters over 28 days and in stimulated field-scale composters over 106 days using protein misfolding cyclic amplification (PMCA). Laboratory-scale composting was conducted using 45 kg of feedlot manure with and without feathers. Compost was mixed at day 14 to generate a second heating cycle, with temperatures ≥55°C occurred for 2 days in the first cycle and less than 1 day in the second cycle. Based on PMCA, PrP<sup>BSE</sup> was reduced by 1 log in compost with and without feathers after 14 days. After 28 days, PrP<sup>BSE</sup> was reduced by 3 logs and between 3–4 logs in compost with and without feathers, respectively. For stimulated field-scale composting, compost piles were constructed using 2,200 kg feedlot manure and mixed at days 44 and 78 to generate three heating cycles. Temperatures ≥55°C were achieved for 67 out of 106 days of composting. However, after 106 days composting reduced PrP<sup>BSE</sup> by only 1–2 logs. Our findings showed that enrichment for proteolytic microorganisms through the addition of feather keratin to compost enhances the degradation of PrP<sup>BSE</sup>. However, other factors such as varying concentrations and strains of PrP<sup>BSE</sup> used and nature of proteolytic microbial populations in compost may cause differential results
<figure><img src="image_25.png" /></figure>

in sheep, chronic wasting disease in white-tailed deer or transmissible mink encephalopathy in cattle were cut at 5 µm thickness. Samples containing the tissue equivalent of as little as one 5 µm section can be used to readily discriminate positive from negative samples using RT-QuIC. The detection approach is avoiding the potential of a mixed chemical/biological waste stream that would occur if organic solvents were used in the removal of paraffin from the same, and offers the diagnostic and research community an addition in the diagnosis of TSEs.

**26. Exploring the structure of mouse prion fibrils by site-directed spin-labelling and ESR spectroscopy**

Kuan Yu Chu<sup>a,b</sup>, Chien Lun Hung<sup>c</sup>, Yun-Wei Chiang<sup>d</sup>, Sunney I. Chan<sup>a,e</sup> and Rita P.-Y. Chen<sup>b,d</sup>

<sup>a</sup>Department of Chemistry, College of Science, Nation Taiwan University, Taipei, Taiwan; <sup>b</sup>Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan; <sup>c</sup>Department of Chemistry, Nation Tsing Hua University, Taipei, Taiwan; <sup>d</sup>Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan; <sup>e</sup>Institute of Chemistry, Academia Sinica, Taipei, Taiwan

CONTACT Kuan Yu Chu brettchu007@gmail.com

**ABSTRACT**

**Introduction:** Recombinant prion protein (PrP) is a 23 kDa soluble protein without GPI anchor and N-glycosylation. The structure of the N-terminal domain is disordered and the structure of the C-terminal domain contains three α-helices, two β-sheets and one disulphide bond connecting helix 2 and 3. To study the structural conversion mechanism of full-length mouse prion protein (mPrP), we explore which segments of the protein are involved in this conformational transition thus form the cross-β structure.

**Methods:** Structure conversion of mPrP was studied using Electron Spin Resonance Spectroscopy (ESR) and Site-Directed Spin Labelling (SDSL). Two intrinsic Cys of mPrP were mutated to Ala. At the selected positions, the residue(s) were replaced by Cys and labelled with MTSSL. The spin-labelled PrPs were seeded by WT PrP fibrils to form various spin-labelled fibrils. The spin-diluted fibrils were prepared by mixing the spin-labelled PrP with WT PrP.

**Results:** ESR results of N174R1/N181R1, N181R1/Q186R1, and T193R1 fibrils showed that the whole helix 2 was converted into several β-strands and acted as part of the amyloid core by forming cross-β structure. For helix 3, not the whole helix 3 is transformed into cross-β structure during fibril formation. The local region around residue 212 might maintain its helicity and connect two intermolecular β-sheets. The 217–224

region formed a β-strand and are involved in the cross-β structure

**Conclusions:** Part of helix 1 of mPrP was unfolded during fibril formation. However, helix 2 and part of helix 3 of mPrP were involved in conformational conversion and formed the cross-β structure. The N-terminal part of helix 3 remained its helical structure.

**27. Biodegradation of BSE prions in compost**

Tim A. McAllister<sup>a</sup>, Shanwei Xu<sup>b</sup>, Sujeema Abeysekara<sup>a,c</sup>, Sandor Dudas<sup>c</sup>, Gordon Mitchell<sup>d</sup> and Stefanie Czub<sup>c</sup>

<sup>a</sup>Agriculture and Agri-Food Canada, Lethbridge Research and Development Centre, Lethbridge, Alberta, Canada; <sup>b</sup>Alberta Agriculture and Forestry, Lethbridge, Alberta, Canada; <sup>c</sup>Canadian and OIE Reference Laboratories for BSE, Canadian Food Inspection Agency Lethbridge Laboratory, Lethbridge, Alberta, Canada; <sup>d</sup>National and OIE Reference Laboratory for Scrapie and CWD, Canadian Food Inspection Agency, Ottawa, Ontario, Canada

CONTACT Tim A. McAllister tim.mcallister@canada.ca

**ABSTRACT**

To reduce the transmission of PrP<sup>BSE</sup>, specified risk material (SRM) that potentially contain PrP<sup>BSE</sup> is required to be removed from the food and feed chains and disposed of via rendering and landfilling or incineration in North America. However, composting could be a more cost effective means of disposing of SRM and be more suitable in areas where SRM pickup services or suitable landfills are unavailable. Our study investigated the degradation of PrP<sup>BSE</sup> (classical BSE) in lab-scale composters over 28 days and in stimulated field-scale composters over 106 days using protein misfolding cyclic amplification (PMCA). Laboratory-scale composting was conducted using 45 kg of feedlot manure with and without feathers. Compost was mixed at day 14 to generate a second heating cycle, with temperatures ≥55°C occurred for 2 days in the first cycle and less than 1 day in the second cycle. Based on PMCA, PrP<sup>BSE</sup> was reduced by 1 log in compost with and without feathers after 14 days. After 28 days, PrP<sup>BSE</sup> was reduced by 3 logs and between 3–4 logs in compost with and without feathers, respectively. For stimulated field-scale composting, compost piles were constructed using 2,200 kg feedlot manure and mixed at days 44 and 78 to generate three heating cycles. Temperatures ≥55°C were achieved for 67 out of 106 days of composting. However, after 106 days composting reduced PrP<sup>BSE</sup> by only 1–2 logs. Our findings showed that enrichment for proteolytic microorganisms through the addition of feather keratin to compost enhances the degradation of PrP<sup>BSE</sup>. However, other factors such as varying concentrations and strains of PrP<sup>BSE</sup> used and nature of proteolytic microbial populations in compost may cause differential results
<figure><img src="image_22.png" /></figure>

in the degradation of PrP<sup>BSE</sup> in lab and field-scale composting systems.

## 28. Optimization of RT-QuIC for detection of seeding activity in preclinical blood samples from prion-infected sheep

Charlotte M. Thomas, Sandra McCutcheon, Richard A. Blanco, Boon Chin Tan and Fiona Houston

The Roslin Institute, R(D)SVS University of Edinburgh, Easter Bush, Edinburgh, Scotland, UK

CONTACT Charlotte M. Thomas Charlotte.Thomas@ed.ac.uk

### ABSTRACT

Recent large-scale surveys of appendix samples estimate that as many as 1 in 2000 people in the UK have abnormal accumulation of disease-associated PrP, suggesting that they may be subclinically infected with variant Creutzfeldt-Jakob disease (vCJD) [1]. If these individuals are infectious, they pose a potential risk to others through blood and organ donation, or contamination of surgical instruments. Indeed, several cases of vCJD have been attributed to receiving blood products from individuals who donated blood during the preclinical stage of the disease [2]. Thus, a robust diagnostic test for vCJD is urgently required to detect these 'silent' (preclinical/subclinical) infections, in order to protect the UK blood supply.

Efforts to develop such a test are complicated by the extremely low concentrations of PrP<sup>Sc</sup> present in readily accessible biological samples, such as blood, and this value is likely to be even lower in preclinical samples. Recently, several platforms have been developed which may be sensitive enough to overcome this challenge. One such technique, an ultrasensitive *in vitro* prion amplification assay, termed 'real-time quaking induced conversion' (RT-QuIC), has already proven to be a highly effective tool in the diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD), when applied to samples of cerebrospinal fluid. [3] However, attempts to develop RT-QuIC into a blood-based diagnostic test for vCJD have been less successful.

Here we present a novel version of the RT-QuIC reaction, using a previously untested recombinant prion protein (recPrP) substrate. Due to a paucity of suitable human samples (which are necessary to optimize the platform and evaluate its performance in detecting preclinical infection), we have optimized our assay using an animal model of vCJD infection - sheep that have been experimentally infected with BSE. We show that our version of the RT-QuIC assay gives positive amplification results when applied to whole blood samples spiked with BSE-infected sheep brain

homogenate. Positive samples are amplified within 20-40 h, with a high degree of analytical sensitivity, equivalent to a 10<sup>-6</sup> dilution of BSE-infected brain homogenate. We then assess the performance of the assay on 'endogenously' infected whole blood samples, collected from BSE-infected sheep at regular time points, from the time of infection to the onset of clinical signs. If this version of the RT-QuIC reaction demonstrates high levels of sensitivity and specificity in identifying blood samples from pre-clinically infected individuals, it could then be adapted for use in humans.

### References

[1] Gill O, Spencer Y, Richard-Loendt A, et al. Prevalent abnormal prion protein in human appendixes after bovine spongiform encephalopathy epizootic: large scale survey. BMJ. 2013;347:f5675.

[2] Peden AH, Head MW, Ritchie DL, et al. Preclinical vCJD after blood transfusion in a PRNP codon 129 heterozygous patient. Lancet 2004;364:527-529.

[3] McGuire LI, Peden AH, Orrú CD, et al. Real time quaking-induced conversion analysis of cerebrospinal fluid in sporadic Creutzfeldt-Jakob disease. Ann Neurol. 2012;72(2):278-285.

## 29. High efficiency detection of all prion subtypes of sporadic Creutzfeldt-Jakob disease by PMCA

Adam Lyon<sup>a</sup>, Sandra Pritzkow<sup>b</sup>, Silvio Notari<sup>b</sup>, Brian Appleby<sup>b</sup>, Pierluigi Gambetti<sup>b</sup> and Claudio Soto<sup>a</sup>

### ABSTRACT

Sporadic Creutzfeldt-Jakob disease (sCJD) is a fatal neurodegenerative disorder representing 85% of human prion diseases. It occurs at a rate of approximately one case per 1,000,000 people per year and its etiology is unknown. There are at least five subtypes of sCJD classified by the methionine (M)/valine (V) polymorphism at codon 129 and by the electrophoretic mobility of the protease-resistant core fragment of PrP<sup>Sc</sup>. The unglycosylated band of PrP<sup>Sc</sup> type 1 is ~21 kDa, and that of PrP<sup>Sc</sup> type 2 is ~19 kDa. The molecular classification aligns with distinct clinical and pathological features. While all subtypes remain invariably fatal, early and accurate diagnosis of sCJD and other human prion diseases is important for patients and their families, as well as for health care providers. Detection by real-time quaking induced conversion (RT-QuIC) of sCJD prions in CSF and from nasal brushings has demonstrated high

<footnote><sup>a</sup>From the Mitchell Center for Alzheimer's Disease and Related Brain Disorders, Department of Neurology, University of Texas Houston Medical School, Houston, Texas, USA; <sup>b</sup>National Prion Disease Pathology Surveillance Center, Case Western University School of Medicine, Cleveland, OH, USA</footnote>
<figure><img src="image_26.png" /></figure>

in the degradation of PrP<sup>BSE</sup> in lab and field-scale composting systems.

**28. Optimization of RT-QuIC for detection of seeding activity in preclinical blood samples from prion-infected sheep**

Charlotte M. Thomas, Sandra McCutcheon, Richard A. Blanco, Boon Chin Tan and Fiona Houston

The Roslin Institute, R(D)SVS University of Edinburgh, Easter Bush, Edinburgh, Scotland, UK

CONTACT Charlotte M. Thomas Charlotte.Thomas@ed.ac.uk

**ABSTRACT**

Recent large-scale surveys of appendix samples estimate that as many as 1 in 2000 people in the UK have abnormal accumulation of disease-associated PrP, suggesting that they may be subclinically infected with variant Creutzfeldt-Jakob disease (vCJD) [1]. If these individuals are infectious, they pose a potential risk to others through blood and organ donation, or contamination of surgical instruments. Indeed, several cases of vCJD have been attributed to receiving blood products from individuals who donated blood during the preclinical stage of the disease [2]. Thus, a robust diagnostic test for vCJD is urgently required to detect these 'silent' (preclinical/subclinical) infections, in order to protect the UK blood supply.

Efforts to develop such a test are complicated by the extremely low concentrations of PrP<sup>Sc</sup> present in readily accessible biological samples, such as blood, and this value is likely to be even lower in preclinical samples. Recently, several platforms have been developed which may be sensitive enough to overcome this challenge. One such technique, an ultrasensitive *in vitro* prion amplification assay, termed 'real-time quaking induced conversion' (RT-QuIC), has already proven to be a highly effective tool in the diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD), when applied to samples of cerebrospinal fluid. [3] However, attempts to develop RT-QuIC into a blood-based diagnostic test for vCJD have been less successful.

Here we present a novel version of the RT-QuIC reaction, using a previously untested recombinant prion protein (recPrP) substrate. Due to a paucity of suitable human samples (which are necessary to optimize the platform and evaluate its performance in detecting preclinical infection), we have optimized our assay using an animal model of vCJD infection – sheep that have been experimentally infected with BSE. We show that our version of the RT-QuIC assay gives positive amplification results when applied to whole blood samples spiked with BSE-infected sheep brain

homogenate. Positive samples are amplified within 20–40 h, with a high degree of analytical sensitivity, equivalent to a 10<sup>-6</sup> dilution of BSE-infected brain homogenate. We then assess the performance of the assay on ‘endogenously’ infected whole blood samples, collected from BSE-infected sheep at regular time points, from the time of infection to the onset of clinical signs. If this version of the RT-QuIC reaction demonstrates high levels of sensitivity and specificity in identifying blood samples from pre-clinically infected individuals, it could then be adapted for use in humans.

**References**

[1] Gill O, Spencer Y, Richard-Loendt A, et al. Prevalent abnormal prion protein in human appendixes after bovine spongiform encephalopathy epizootic: large scale survey. BMJ. 2013;347:f5675.

[2] Peden AH, Head MW, Ritchie DL, et al. Preclinical vCJD after blood transfusion in a PRNP codon 129 heterozygous patient. Lancet 2004;364:527–529.

[3] McGuire LI, Peden AH, Orrú CD, et al. Real time quaking-induced conversion analysis of cerebrospinal fluid in sporadic Creutzfeldt-Jakob disease. Ann Neurol. 2012;72(2):278–285.

**29. High efficiency detection of all prion subtypes of sporadic Creutzfeldt-Jakob disease by PMCA**

Adam Lyon<sup>a</sup>, Sandra Pritzkow<sup>b</sup>, Silvio Notari<sup>b</sup>, Brian Appleby<sup>b</sup>, Pierluigi Gambetti<sup>b</sup> and Claudio Soto<sup>a</sup>

**ABSTRACT**

Sporadic Creutzfeldt-Jakob disease (sCJD) is a fatal neurodegenerative disorder representing 85% of human prion diseases. It occurs at a rate of approximately one case per 1,000,000 people per year and its etiology is unknown. There are at least five subtypes of sCJD classified by the methionine (M)/valine (V) polymorphism at codon 129 and by the electrophoretic mobility of the protease-resistant core fragment of PrP<sup>Sc</sup>. The unglycosylated band of PrP<sup>Sc</sup> type 1 is ~21 kDa, and that of PrP<sup>Sc</sup> type 2 is ~19 kDa. The molecular classification aligns with distinct clinical and pathological features. While all subtypes remain invariably fatal, early and accurate diagnosis of sCJD and other human prion diseases is important for patients and their families, as well as for health care providers. Detection by real-time quaking induced conversion (RT-QuIC) of sCJD prions in CSF and from nasal brushings has demonstrated high

<footnote><sup>a</sup>From the Mitchell Center for Alzheimer's Disease and Related Brain Disorders, Department of Neurology, University of Texas Houston Medical School, Houston, Texas, USA; <sup>b</sup>National Prion Disease Pathology Surveillance Center, Case Western University School of Medicine, Cleveland, OH, USA</footnote>
<figure><img src="image_23.png" /></figure>

sensitivity and specificity, but the challenge remains to detect sCJD prions in fluids and tissues whose collection is less invasive. Previously, we have shown successful detection of variant CJD prions in blood and urine by protein misfolding cyclic amplification (PMCA). The main goal of this study was to adapt the PMCA assay for high sensitive detection of all forms of sCJD to allow type identification of sCJD PrPSc in patients' blood and urine. Our results demonstrate efficient amplification of all sCJD subtypes by PMCA in brain homogenate, with a variable rate depending on the specific strain. Indeed, we can reproducibly detect at least a 10<sup>-7</sup>-10<sup>-11</sup> dilution of brain homogenate from all samples analysed. Currently, we are analysing blood and urine samples from patients with sCJD with diverse genotypes. This is a crucial step towards implementing PMCA as a general tool for routine diagnosis of sCJD including sCJD subtype, and to screen blood to increase the safety of the blood supply.

## Funding

SB1NS079060, R01 NS083687, P01 AI106705 and Charles S. Britton Fund (PG).

## 30. A blue light optogenetic and CRISPR/Cas9 system for inducible PrPC expression

Luis A. Arce<sup>a,b</sup>, Serene Wohlgemuth<sup>a</sup> and David Westaway<sup>a,b</sup>

CONTACT Luis A. Arce larce@ualberta.ca

## ABSTRACT

An inducible system for expression of wild type PrPC can offer advantages for investigating the chemical biology of this GPI-linked cell-surface protein. Previous inducible PrPC systems have utilized non-physiological xenobiotic compounds such as tetracycline to control gene expression [1,2]. However, wishing to avoid off-target chemical effects of xenobiotic additives, we are developing an inducible system controlled by light. CRISPR/Cas9 systems that activate transcription (CRISPRa) utilize transcriptional activation domains (TAD), as found in VP64 or p65 proteins, attached to a modified Cas9 (called dCas9) [3]. dCas9 has inactivated endonuclease function but can still be directed to a specific genomic locus by a 'guide RNA' (gRNA); previous studies have exploited this interaction by the development of dCas9/TAD fusion proteins that allow for the activation and overexpression of targeted genes [4]. Inducible CRISPR-based systems have been developed typically using a split-Cas9 method and small effector molecules, such as rapamycin. However, these systems

can also exhibit leakiness [5]. An alternative system which utilizes the light inducible protein interactions between cryptochrome 2 (CRY2) and CIB1 from *Arabidopsis thaliana* has proven to be effective in both gene induction and mitigation of residual transcriptional activity [6,7]. The system contains two fusion proteins; the 'anchor' (dCas9/CIB1), and the 'activator' (CRY2/TAD); targeting of dCas9 is achieved by the gRNA and then heterodimerization of CRY2 and CIB1 is based upon blue light irradiation. Here we describe adaptation of this optogenetic system to modify transcription initiated from the human *Prnp* promoter region. Following a gRNA design phase, corresponding sequences were inserted into an expression vector. Serial transfections were used to deliver the two fusion proteins plasmids and designed gRNA plasmids into the human HAP1 cell line. Transfected cells are subjected to blue light irradiation and then assessed for PrPC<sup>C</sup> levels using quantitative capillary westerns. This inducible system may offer advantages for drug screening to identify novel chemicals that modify PrPC<sup>B</sup> biology.

**KEYWORDS:** CRISPR Cas9; optogenetics; PrPC<sup>C</sup>; drug screen

## References

[1] S. Huang, et al. PNAS 2007;104(16):6800-6805.

[2] E. Sabuncu, et al. Biochem Biophys Res Commun 2005;337(3):791-798.

[3] L. A. Gilbert, et al. Cell 2013;154(2):442-451.

[4] M. E. Tanenbaum, et al. Cell 2014; 159(3):635-646

[5] B. Zetsche, et al. Nat Biotechnol 2015;33(2):139-141.

[6] M. J. Kennedy, et al. Nat Methods 2010;7(12):973-975.

[7] Y. Nihongaki, et al. Chem Biol 2015;22(2):169-174.

## 31. Exploring the role of PrP in axonal tract formation during zebrafish nervous system development

Fabrizio Cuevas<sup>a,b</sup>, Ignacio Gutierrez<sup>a,b</sup>, Carmen G. Lemus<sup>a,b</sup> and Miguel L. Concha<sup>a,b,c</sup>

CONTACT Fabrizio Cuevas fabriziocuevas@gmail.com

## ABSTRACT

Although the role of PrPc in neurite outgrowth, neuronal differentiation and synapse formation has been described in vitro, little is known of this phenomenon in vivo and especially during development. Zebrafish (Danio rerio) has emerged as a promising model to study PrP physiological function because of its external development, embryo

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

<footnote><sup>a</sup>Anatomy and Developmental Biology, Institute of Biomedical Sciences, Faculty of Medicine, Universidad de Chile, Santiago, Chile; <sup>b</sup>Biomedical Neuroscience Institute, Santiago, Chile; <sup>c</sup>Center for Geroscience, Brain Health and Metabolism, Santiago, Chile</footnote>
<figure><img src="image_27.png" /></figure>

sensitivity and specificity, but the challenge remains to detect sCJD prions in fluids and tissues whose collection is less invasive. Previously, we have shown successful detection of variant CJD prions in blood and urine by protein misfolding cyclic amplification (PMCA). The main goal of this study was to adapt the PMCA assay for high sensitive detection of all forms of sCJD to allow type identification of sCJD PrPSc in patients' blood and urine. Our results demonstrate efficient amplification of all sCJD subtypes by PMCA in brain homogenate, with a variable rate depending on the specific strain. Indeed, we can reproducibly detect at least a 10<sup>-7</sup>–10<sup>-11</sup> dilution of brain homogenate from all samples analysed. Currently, we are analysing blood and urine samples from patients with sCJD with diverse genotypes. This is a crucial step towards implementing PMCA as a general tool for routine diagnosis of sCJD including sCJD subtype, and to screen blood to increase the safety of the blood supply.

## Funding

SB1NS079060, R01 NS083687, P01 AI106705 and Charles S. Britton Fund (PG).

# 30. A blue light optogenetic and CRISPR/Cas9 system for inducible PrPC expression

Luis A. Arce<sup>a,b</sup>, Serene Wohlgemuth<sup>a</sup> and David Westaway<sup>a,b</sup>

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

CONTACT Luis A. Arce larce@ualberta.ca

## ABSTRACT

An inducible system for expression of wild type PrPC can offer advantages for investigating the chemical biology of this GPI-linked cell-surface protein. Previous inducible PrPC systems have utilized non-physiological xenobiotic compounds such as tetracycline to control gene expression [1,2]. However, wishing to avoid off-target chemical effects of xenobiotic additives, we are developing an inducible system controlled by light. CRISPR/Cas9 systems that activate transcription (CRISPRa) utilize transcriptional activation domains (TAD), as found in VP64 or p65 proteins, attached to a modified Cas9 (called dCas9) [3]. dCas9 has inactivated endonuclease function but can still be directed to a specific genomic locus by a 'guide RNA' (gRNA); previous studies have exploited this interaction by the development of dCas9/TAD fusion proteins that allow for the activation and overexpression of targeted genes [4]. Inducible CRISPR-based systems have been developed typically using a split-Cas9 method and small effector molecules, such as rapamycin. However, these systems

can also exhibit leakiness [5]. An alternative system which utilizes the light inducible protein interactions between cryptochrome 2 (CRY2) and CIB1 from *Arabidopsis thaliana* has proven to be effective in both gene induction and mitigation of residual transcriptional activity [6,7]. The system contains two fusion proteins; the ‘anchor’ (dCas9/ CIB1), and the ‘activator’ (CRY2/TAD); targeting of dCas9 is achieved by the gRNA and then heterodimerization of CRY2 and CIB1 is based upon blue light irradiation. Here we describe adaptation of this optogenetic system to modify transcription initiated from the human *Prnp* promoter region. Following a gRNA design phase, corresponding sequences were inserted into an expression vector. Serial transfections were used to deliver the two fusion proteins plasmids and designed gRNA plasmids into the human HAP1 cell line. Transfected cells are subjected to blue light irradiation and then assessed for PrPC<sup>C</sup> levels using quantitative capillary westerns. This inducible system may offer advantages for drug screening to identify novel chemicals that modify PrPC<sup>C</sup> biology.

**KEYWORDS:** CRISPR Cas9; optogenetics; PrPC<sup>C</sup>; drug screen

## References

[1] S. Huang, et al. PNAS 2007;104(16):6800–6805.

[2] E. Sabuncu, et al. Biochem Biophys Res Commun 2005;337(3):791–798.

[3] L. A. Gilbert, et al. Cell 2013;154(2):442–451.

[4] M. E. Tanenbaum, et al. Cell 2014; 159(3):635–646

[5] B. Zetsche, et al. Nat Biotechnol 2015;33(2):139–141.

[6] M. J. Kennedy, et al. Nat Methods 2010;7(12):973–975.

[7] Y. Nihongaki, et al. Chem Biol 2015;22(2):169–174.

# 31. Exploring the role of PrP in axonal tract formation during zebrafish nervous system development

Fabrizio Cuevas<sup>a,b</sup>, Ignacio Gutierrez<sup>a,b</sup>, Carmen G. Lemus<sup>a,b</sup> and Miguel L. Concha<sup>a,b,c</sup>

<sup>a</sup>Anatomy and Developmental Biology, Institute of Biomedical Sciences, Faculty of Medicine, Universidad de Chile, Santiago, Chile; <sup>b</sup>Biomedical Neuroscience Institute, Santiago, Chile; <sup>c</sup>Center for Geroscience, Brain Health and Metabolism, Santiago, Chile

CONTACT Fabrizio Cuevas fabriziocuevas@gmail.com

## ABSTRACT

Although the role of PrPc in neurite outgrowth, neuronal differentiation and synapse formation has been described *in vitro*, little is known of this phenomenon *in vivo* and especially during development. Zebrafish (Danio rerio) has emerged as a promising model to study PrP physiological function because of its external development, embryo
<figure><img src="image_24.png" /></figure>

transparency and amenability to genetic manipulation. The developmental expression of genes encoding for the zebrafish PrP paralogs prp-1 and prp-2 is still controversial thus we first revisited in detail the spatiotemporal expression of both genes by *in situ* hybridization. prp2 is expressed from 18 h post fertilization (hpf) in the encephalon and the first segments of the spinal cord, and also in structures outside the nervous system. In contrast, prp1 is detectable in the central nervous system only from 48 hpf, and in cranial ganglia from 60 hpf. These results are consistent with a previous report [1], but differ in the lack of expression seen at earlier stages. To begin exploring the function of PrP in axonal tract formation we perfomed acetylated tubulin by indirect immunofluorescence in mutant embryos and larvae lacking *prp-1* and *prp-2* function. We found that prp-1 and prp-2 mutants both show weaker intensity of anti-acetylated tubulin staining and less axonal arborization in some specific brain regions, like the olfactory bulb and cranial ganglia, and thinner tracts at the level of the cerebellar commissure. The presence of abnormal tracts in prp-1 and prp-2 mutants is intriguing given the normal appearance of embryos and larvae. We are currently dissecting the axonal tract deficiencies using fluorescent reporters in prp-1 and prp-2 mutants.

## Funding

CONICYT PFCHA/DOCTORADO BECAS CHILE/ 2017-21,171,130, ICM P09-015-F and Fondap 15,150,012.

## Reference

[1] Cotto E, Andre M, Forgue J, et al. Molecular characterization, phylogenetic relationships, and developmental expression patterns of prion genes in zebrafish (Danio rerio). *Febs J.* 2005;272(2):500-13.

## 32. The EV-export pathway for misfolded proteins

Desmond Pinkª, John Lewisª,ᵇ and Janice E.A. Braunᶜ

ªNanostics Precision Health, Edmonton, Alberta, Canada; ᵇHotchkiss Brain Institute, Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada; ᶜDepartment of Oncology, University of Alberta, Edmonton, Alberta, Canada

CONTACT Janice E.A. Braun braunj@ucalgary.ca

## ABSTRACT

Extracellular vesicles (EVs) are a collection of secreted vesicles of diverse size and cargo that are implicated in the physiological removal of nonfunctional proteins as well as the cell-to-cell transmission of disease-causing-proteins in several neurodegenerative diseases. We have shown that the molecular chaperone, cysteine

string protein (CSPα; DnaJC5), is responsible for the export of disease-causing-misfolded proteins from neurons via EVs. We report here that CSPα-EVs efficiently deliver GFP-tagged 72Q huntingtin<sup>exon1</sup> to naive neurons. When we analysed the heterogeneous EV pool, we found that the misfolded GFP-tagged 72Q huntingtin<sup>exon1</sup> cargo was primarily found in EVs between 180-240nm. We further determined that cargo-loading of GFP-tagged 72Q huntingtin<sup>exon1</sup> into EVs was impaired by resveratrol. In addition to CSPα, we identified two other J protein co-chaperones, DnaJB2 and DnaJB6 that facilitate EV export of GFP-tagged 72Q huntingtin<sup>exon1</sup>. While human mutations in CSPα cause the neurodegenerative disorder, adult neuronal ceroid lipofuscinosis, mutations in DnaJB6 cause limb-girdle muscular dystrophy and mutations in DnaJB2 are linked to the neurodegenerative disorders, Charcot Marie Tooth disease, distal hereditary motor neuropathy, spinal muscular atrophy and juvenile Parkinsonism. Our data provides new insights into the parallels between proteostasis and EV export, as three J proteins linked to disease in humans are the same as those that mediate EV genesis and export of misfolded proteins.

## 33. Detection of CWD prions in third eyelids of deer and elk

Sarah K. Cooperª, Clare E. Hooverª,ᵇ, Davin M. Hendersonª, Nathaniel D. Denkersª, Candace K. Mathiasonª and Edward A. Hooverª

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

CONTACT Sarah K. Cooper scooper2@rams.colostate.edu

## ABSTRACT

**Background:** The increasing prevalence of CWD globally, makes critical the development of fast, cost-effective methods to detect the disease in hunter-harvested deer and assist wildlife population disease management. Based in part on the demonstration of PrPSc in the third eyelid lymphoid follicle in sheep with scrapie, we explored the third eyelid for its potential as a non-invasive and easily accessible lymphoid tissue that could be sampled without special anatomical training antemortem and postmortem for PrPC<sup>WD</sup> detection by real-time quaking induced conversion (RT-QuIC) and immunohistochemistry (IHC).

**Methods:** We compared RT-QuIC detection sensitivity in the third eyelid to the retropharyngeal lymph node and obex region of the brain from experimentally CWD inoculated white-tailed deer and naturally occurring subclinical
<figure><img src="image_28.png" /></figure>

transparency and amenability to genetic manipulation. The developmental expression of genes encoding for the zebrafish PrP paralogs prp-1 and prp-2 is still controversial thus we first revisited in detail the spatiotemporal expression of both genes by *in situ* hybridization. prp2 is expressed from 18 h post fertilization (hpf) in the encephalon and the first segments of the spinal cord, and also in structures outside the nervous system. In contrast, prp1 is detectable in the central nervous system only from 48 hpf, and in cranial ganglia from 60 hpf. These results are consistent with a previous report [1], but differ in the lack of expression seen at earlier stages. To begin exploring the function of PrP in axonal tract formation we perfomed acetylated tubulin by indirect immunofluorescence in mutant embryos and larvae lacking *prp-1* and *prp-2* function. We found that prp-1 and prp-2 mutants both show weaker intensity of anti-acetylated tubulin staining and less axonal arborization in some specific brain regions, like the olfactory bulb and cranial ganglia, and thinner tracts at the level of the cerebellar commissure. The presence of abnormal tracts in prp-1 and prp-2 mutants is intriguing given the normal appearance of embryos and larvae. We are currently dissecting the axonal tract deficiencies using fluorescent reporters in prp-1 and prp-2 mutants.

## Funding

CONICYT PFCHA/DOCTORADO BECAS CHILE/ 2017-21,171,130, ICM P09-015-F and Fondap 15,150,012.

## Reference

[1] Cotto E, Andre M, Forgue J, et al. Molecular characterization, phylogenetic relationships, and developmental expression patterns of prion genes in zebrafish (Danio rerio). *Febs J.* 2005;272(2):500-13.

## 32. The EV-export pathway for misfolded proteins

Desmond Pinkª, John Lewisª,ᵇ and Janice E.A. Braunᶜ

ªNanostics Precision Health, Edmonton, Alberta, Canada; ᵇHotchkiss Brain Institute, Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada; ᶜDepartment of Oncology, University of Alberta, Edmonton, Alberta, Canada

CONTACT Janice E.A. Braun braunj@ucalgary.ca

## ABSTRACT

Extracellular vesicles (EVs) are a collection of secreted vesicles of diverse size and cargo that are implicated in the physiological removal of nonfunctional proteins as well as the cell-to-cell transmission of disease-causing-proteins in several neurodegenerative diseases. We have shown that the molecular chaperone, cysteine

string protein (CSPα; DnaJC5), is responsible for the export of disease-causing-misfolded proteins from neurons via EVs. We report here that CSPα-EVs efficiently deliver GFP-tagged 72Q huntingtin<sup>exon1</sup> to naive neurons. When we analysed the heterogeneous EV pool, we found that the misfolded GFP-tagged 72Q huntingtin<sup>exon1</sup> cargo was primarily found in EVs between 180-240nm. We further determined that cargo-loading of GFP-tagged 72Q huntingtin<sup>exon1</sup> into EVs was impaired by resveratrol. In addition to CSPα, we identified two other J protein co-chaperones, DnaJB2 and DnaJB6 that facilitate EV export of GFP-tagged 72Q huntingtin<sup>exon1</sup>. While human mutations in CSPα cause the neurodegenerative disorder, adult neuronal ceroid lipofuscinosis, mutations in DnaJB6 cause limb-girdle muscular dystrophy and mutations in DnaJB2 are linked to the neurodegenerative disorders, Charcot Marie Tooth disease, distal hereditary motor neuropathy, spinal muscular atrophy and juvenile Parkinsonism. Our data provides new insights into the parallels between proteostasis and EV export, as three J proteins linked to disease in humans are the same as those that mediate EV genesis and export of misfolded proteins.

## 33. Detection of CWD prions in third eyelids of deer and elk

Sarah K. Cooperª, Clare E. Hooverª,ᵇ, Davin M. Hendersonª, Nathaniel D. Denkersª, Candace K. Mathiasonª and Edward A. Hooverª

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

CONTACT Sarah K. Cooper scooper2@rams.colostate.edu

## ABSTRACT

**Background:** The increasing prevalence of CWD globally, makes critical the development of fast, cost-effective methods to detect the disease in hunter-harvested deer and assist wildlife population disease management. Based in part on the demonstration of PrPSc in the third eyelid lymphoid follicle in sheep with scrapie, we explored the third eyelid for its potential as a non-invasive and easily accessible lymphoid tissue that could be sampled without special anatomical training antemortem and postmortem for PrPC<sup>WD</sup> detection by real-time quaking induced conversion (RT-QuIC) and immunohistochemistry (IHC).

**Methods:** We compared RT-QuIC detection sensitivity in the third eyelid to the retropharyngeal lymph node and obex region of the brain from experimentally CWD inoculated white-tailed deer and naturally occurring subclinical
<figure><img src="image_25.png" /></figure>

elk using IHC. We examined symptomatic and asymptomatic white-tailed deer inoculated with CWD(+) and CWD(-) brain homogenate or saliva via the *per os* or aerosol route. In addition, we examined samples from asymptomatic, naturally exposed elk which were culled due to being RAMALT biopsy positive or suspect by RT-QuIC.

**Results:** We identified prion seeding activity in third eyelids in 24 out of 25 (96%) samples from terminal, experimentally CWD-infected deer and found RT-QuIC positivity in the third eyelid as early as 1 month after experimental exposure to CWD. In addition, we identified prion seeding activity in third eyelids in 17 out of 25 (68%) samples asymptomatic, naturally exposed elk. By contrast, IHC detected prion deposition in third eyelid lymphoid follicles in 5 of 10 deer (50%). We show that RT-QuIC can be more sensitive for CWD prion detection in the third eyelid compared to IHC.

**Conclusions:** RT-QuIC testing on the third eyelid is a rapid and sensitive means for post-mortem detection of CWD with potential to augment CWD diagnostics and hunter testing compliance.

## Funding

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

## 34. microRNAs profiling in patient bloods identifies a novel signature associated with sCJD disease progression

Penny Norsworthy, John Collinge, Simon Mead and
Emmanuelle Viré

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

CONTACT Emmanuelle Viré e.vire@ucl.ac.uk

## ABSTRACT

Prion diseases are fatal neurodegenerative diseases of humans and animals caused by the misfolding and aggregation of prion protein (PrP). Misfolded proteins play an important role in many forms of dementia but despite intensive efforts to better understand the pathophysiology of these diseases, the mechanisms involved are still poorly understood. Although neurodegeneration is affecting millions of people worldwide there is a huge unmet need for novel diagnostic and treatment approaches in neurodegenerative disorders to counter therapeutic nihilism in this area. Recent studies have implicated a role for genetic and epigenetic variation in neurodegenerative disorders. To date, however, no study has systematically explored the contribution of epigenetic changes in human prion diseases.

microRNAs (miRNAs) play crucial role in human diseases, and the discovery of the presence of cell-free miRNAs in the blood stream probed researchers to investigate the role of miRNAs as potential non-invasive biomarkers.

We performed genome-wide microRNA deep sequencing on 107 blood samples from patients with sporadic CJD and healthy controls and identified five differentially expressed microRNAs. We confirmed that this miRNA signature is specific to sporadic CJD since the level of those 5 miRNA remained unchanged in other neurodegenerative disorders (CJD and Alzheimer's diseases). Investigating longitudinal samples from more than 20 patients with sCJD, we found that the miRNA signature strongly correlates with disease severity. These results have been replicated using both an independent cohort and qPCR technology.

Integrating our data, we have established a sCJD specific signature, with genes between the two groups being enriched in functions related to neuroinflammation and immunity; and have correlated it with age of onset, disease progression, severity, decline, and genotype.

In light of our findings, we will discuss the potential implications for this novel gene signature to complement existing non-invasive biomarkers and its relevance for disease management.

## 35. Process of structural diversification and synergy between prion subassemblies during host adaptation

Angélique Igel-Egalonª, Mohammed Moudjouª, Florent Lafërrièreª, Philippe Tixadorª, Jan Bohlª, Mathieu Mezacheª,ᵇ, Tina Knäppleª, Laetitia Herzogª, Fabienne Reineª, Marie Doumicᵇ, Vincent Béringueª and Human Rezaeiª

ªVIM, INRA, Université Paris-Saclay, Jouy-en-Josas, France; ᵇINRIA, MAMBA, Université Paris VI, Paris, France

CONTACT Human Rezaei human.rezaei@inra.fr

## ABSTRACT

In prion diseases, the cellular prion protein PrPC can misfold into PrPSc and auto-organize into conformationally distinct assemblies or strains. A plethora of observations reports the existence of PrPSc structural heterogeneity within prion strains, suggesting the emergence and coevolution of structurally distinct PrPSc subassemblies during prion replication in controlled environment. However, the nature of and the underlying processes of the structural diversification remain poorly explored and mainly based on conjectural observations. Conceptually, such structural heterogeneity appears
<figure><img src="image_29.png" /></figure>

elk using IHC. We examined symptomatic and asymptomatic white-tailed deer inoculated with CWD(+) and CWD(-) brain homogenate or saliva via the *per os* or aerosol route. In addition, we examined samples from asymptomatic, naturally exposed elk which were culled due to being RAMALT biopsy positive or suspect by RT-QuIC.

**Results:** We identified prion seeding activity in third eyelids in 24 out of 25 (96%) samples from terminal, experimentally CWD-infected deer and found RT-QuIC positivity in the third eyelid as early as 1 month after experimental exposure to CWD. In addition, we identified prion seeding activity in third eyelids in 17 out of 25 (68%) samples asymptomatic, naturally exposed elk. By contrast, IHC detected prion deposition in third eyelid lymphoid follicles in 5 of 10 deer (50%). We show that RT-QuIC can be more sensitive for CWD prion detection in the third eyelid compared to IHC.

**Conclusions:** RT-QuIC testing on the third eyelid is a rapid and sensitive means for post-mortem detection of CWD with potential to augment CWD diagnostics and hunter testing compliance.

## Funding

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

## 34. microRNAs profiling in patient bloods identifies a novel signature associated with sCJD disease progression

Penny Norsworthy, John Collinge, Simon Mead and
Emmanuelle Viré

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

CONTACT Emmanuelle Viré e.vire@ucl.ac.uk

## ABSTRACT

Prion diseases are fatal neurodegenerative diseases of humans and animals caused by the misfolding and aggregation of prion protein (PrP). Misfolded proteins play an important role in many forms of dementia but despite intensive efforts to better understand the pathophysiology of these diseases, the mechanisms involved are still poorly understood. Although neurodegeneration is affecting millions of people worldwide there is a huge unmet need for novel diagnostic and treatment approaches in neurodegenerative disorders to counter therapeutic nihilism in this area. Recent studies have implicated a role for genetic and epigenetic variation in neurodegenerative disorders. To date, however, no study has systematically explored the contribution of epigenetic changes in human prion diseases.

microRNAs (miRNAs) play crucial role in human diseases, and the discovery of the presence of cell-free miRNAs in the blood stream probed researchers to investigate the role of miRNAs as potential non-invasive biomarkers.

We performed genome-wide microRNA deep sequencing on 107 blood samples from patients with sporadic CJD and healthy controls and identified five differentially expressed microRNAs. We confirmed that this miRNA signature is specific to sporadic CJD since the level of those 5 miRNA remained unchanged in other neurodegenerative disorders (CJD and Alzheimer's diseases). Investigating longitudinal samples from more than 20 patients with sCJD, we found that the miRNA signature strongly correlates with disease severity. These results have been replicated using both an independent cohort and qPCR technology.

Integrating our data, we have established a sCJD specific signature, with genes between the two groups being enriched in functions related to neuroinflammation and immunity; and have correlated it with age of onset, disease progression, severity, decline, and genotype.

In light of our findings, we will discuss the potential implications for this novel gene signature to complement existing non-invasive biomarkers and its relevance for disease management.

## 35. Process of structural diversification and synergy between prion subassemblies during host adaptation

Angélique Igel-Egalonª, Mohammed Moudjouª, Florent Laferrrièreª, Philippe Tixadorª, Jan Bohlª, Mathieu Mezacheª,ᵇ, Tina Knäppleª, Laetitia Herzogª, Fabienne Reineª, Marie Doumicᵇ, Vincent Béringueª and Human Rezaeiª

ªVIM, INRA, Université Paris-Saclay, Jouy-en-Josas, France; ᵇINRIA, MAMBA, Université Paris VI, Paris, France

CONTACT Human Rezaei human.rezaei@inra.fr

## ABSTRACT

In prion diseases, the cellular prion protein PrPC can misfold into PrPSc and auto-organize into conformationally distinct assemblies or strains. A plethora of observations reports the existence of PrPSc structural heterogeneity within prion strains, suggesting the emergence and coevolution of structurally distinct PrPSc subassemblies during prion replication in controlled environment. However, the nature of and the underlying processes of the structural diversification remain poorly explored and mainly based on conjectural observations. Conceptually, such structural heterogeneity appears
central to prion host adaptation since the actual
paradigm consists mostly on the best replicator
selection, i.e. selection of the best-fit subset of
PrPSc assemblies within the PrPSc structural cloud.

Here, we first characterized the evolution of the PrPSc quaternary structure in early versus late stage of prion replication (in a homotypic PrP context) *in vivo* and in bona fide cell-free amplification assays. Regardless of the strain studied, the early replication stage conduced to the preferential formation of small PrPSc oligomers, thus highlighting a quaternary structural convergence phenomenon. The evolution of these small oligomeric assemblies led to the formation of structurally different multimers through a secondary templating pathway in an autocatalytic and PrPC-dependent manner. Second, we addressed the role of such structural PrPSc diversification in prion adaptation on cross-species transmission. We isolated by size-fractionation or by dilution the formed PrPSc assemblies and measured their relative propensity to cross an existing species barrier, as modelled by transgenic mice expressing heterotypic PrPC. We showed impaired and aberrant fitness of the isolated PrPSc subassemblies as compared to the unfractionated and undiluted populations, respectively.

Altogether our observations uncover the existence of
two mechanistic processes during prion replication:
first, prion structural diversification occurs through a
secondary templating pathway. Second, overcoming
species barriers requires complementation between the
PrPSc subassemblies. These processes explain how
structurally diverse assemblies can coexist despite com-
petition for the same PrPC substrate and truly chal-
lenge the best- replicator selection models.

36. *Structural characterization of amyloidogenic proteins implicated in neurodegeneration under conditions of intracellular crowding and in non-polar environments*

Nikolay Blinov<sup>a,b</sup>, David S. Wishart<sup>c</sup>, Neil R. Cashman<sup>d</sup>,
and Andriy Kovalenko<sup>a,b</sup>

CONTACT Nikolay Blinov nblinov@ualberta.ca

ABSTRACT

Misfolding and aggregation of proteins is a key patho-
logical event in many neurodegenerative disorders,
including the prion diseases, Alzheimer's disease
(AD), and Amyotrophic Lateral Sclerosis. Structural

properties of proteins and their aggregates (oligomers
and amyloid fibrils) are essentially affected by their
solvation environments, the presence of metabolites
and salt ions. Structural characterization of oligomers
which may be the most toxic agents in neurodegenera-
tion is a very important task because the inhibition of
formation of these aggregates and their pathological
action has been proposed as a therapeutic target to
fight neurodegeneration.

Experimental structural studies are complicated by
the transient nature of oligomers. In the case of AD,
amyloid (A)β peptides (constituent units of the neuro-
toxic oligomers responsible for neurotoxicity in AD) are
intrinsically disordered, which introduces another level
of complexity to the problem. Despite the significant
efforts, there is no consensus on structure and aggrega-
tion pathways of Aβ peptides in cellular environments.
The situation is further complicated by the fact that the
peptides can aggregate and perform their neurotoxic
activity intracellularly. The intracellular Aβ species can
also be secreted into the extracellular space where they
may trigger prion-like pathological misfolding.

In this poster, we provide an overview of our recent
efforts towards structural characterization of the amyloido-
genic proteins and their aggregates under different solvent
environments. The new methodology has been developed
based on the 3D-RISM-KH molecular theory of solvation
to account for the effects of specific conditions of intra and
extracellular environments on the conformational equili-
brium of intrinsically disordered peptides. Applied to AD,
it shows the similarities and differences between conforma-
tional properties of Aβ peptides under conditions of intra-
cellular crowding and in non-polar environments (such as
imposed by the neuronal lipid membrane). Specifically,
both the crowding and the non-polar solvation make the
peptides less disordered, which may result in the increase
of their propensity for aggregation. Among the differences
are the opposite trends in bend and turn secondary struc-
ture contents under different conditions. We also show
that the 3D-RISM-KH based methodology could be used
to predict effects of synaptic metabolites and other small
molecules (e.g. therapeutic agents) on the structure of the
peptides. This feature of the methodology can be used to
screen potential inhibitors of pathological misfolding of
proteins in neurodegeneration.

37. *Spontaneous neurodegeneration in transgenic mice caused by histidine to tyrosine substitution at non-OR copper-binding site*

Alba Marín-Moreno<sup>a*</sup>, Thanh Hoa Tran<sup>b*</sup>, Juan Carlos Espinosa<sup>a</sup>, Edoardo Bistaffa<sup>c</sup>, Fabio Moda<sup>c</sup>, Juan María Torres<sup>a</sup> and Giuseppe Legname<sup>b</sup>

<footnote><sup>a</sup>University of Alberta, Department of Mechanical Engineering, Edmonton, Canada; <sup>b</sup>NRC Nanotechnology Research Centre, Edmonton, Canada; <sup>c</sup>University of Alberta, Departments of Biological Sciences and Computing Science, Edmonton, Canada; <sup>d</sup>University of British Columbia, Department of Medicine, Vancouver, Canada</footnote>
central to prion host adaptation since the actual
paradigm consists mostly on the best replicator
selection, i.e. selection of the best-fit subset of
PrPSc assemblies within the PrPSc structural cloud.

Here, we first characterized the evolution of the PrPSc quaternary structure in early versus late stage of prion replication (in a homotypic PrP context) *in vivo* and in bona fide cell-free amplification assays. Regardless of the strain studied, the early replication stage conduced to the preferential formation of small PrPSc oligomers, thus highlighting a quaternary structural convergence phenomenon. The evolution of these small oligomeric assemblies led to the formation of structurally different multimers through a secondary templating pathway in an autocatalytic and PrPC-dependent manner. Second, we addressed the role of such structural PrPSc diversification in prion adaptation on cross-species transmission. We isolated by size-fractionation or by dilution the formed PrPSc assemblies and measured their relative propensity to cross an existing species barrier, as modelled by transgenic mice expressing heterotypic PrPC. We showed impaired and aberrant fitness of the isolated PrPSc subassemblies as compared to the unfractionated and undiluted populations, respectively.

Altogether our observations uncover the existence of
two mechanistic processes during prion replication:
first, prion structural diversification occurs through a
secondary templating pathway. Second, overcoming
species barriers requires complementation between the
PrPSc subassemblies. These processes explain how
structurally diverse assemblies can coexist despite com-
petition for the same PrPC substrate and truly chal-
lenge the best- replicator selection models.

36. *Structural characterization of amyloidogenic proteins implicated in neurodegeneration under conditions of intracellular crowding and in non-polar environments*

Nikolay Blinov<sup>a,b</sup>, David S. Wishart<sup>c</sup>, Neil R. Cashman<sup>d</sup>,
and Andriy Kovalenko<sup>a,b</sup>

CONTACT Nikolay Blinov nblinov@ualberta.ca

ABSTRACT

Misfolding and aggregation of proteins is a key patho-
logical event in many neurodegenerative disorders,
including the prion diseases, Alzheimer's disease
(AD), and Amyotrophic Lateral Sclerosis. Structural

properties of proteins and their aggregates (oligomers
and amyloid fibrils) are essentially affected by their
solvation environments, the presence of metabolites
and salt ions. Structural characterization of oligomers
which may be the most toxic agents in neurodegenera-
tion is a very important task because the inhibition of
formation of these aggregates and their pathological
action has been proposed as a therapeutic target to
fight neurodegeneration.

Experimental structural studies are complicated by
the transient nature of oligomers. In the case of AD,
amyloid (A)β peptides (constituent units of the neuro-
toxic oligomers responsible for neurotoxicity in AD) are
intrinsically disordered, which introduces another level
of complexity to the problem. Despite the significant
efforts, there is no consensus on structure and aggrega-
tion pathways of Aβ peptides in cellular environments.
The situation is further complicated by the fact that the
peptides can aggregate and perform their neurotoxic
activity intracellularly. The intracellular Aβ species can
also be secreted into the extracellular space where they
may trigger prion-like pathological misfolding.

In this poster, we provide an overview of our recent
efforts towards structural characterization of the amyloido-
genic proteins and their aggregates under different solvent
environments. The new methodology has been developed
based on the 3D-RISM-KH molecular theory of solvation
to account for the effects of specific conditions of intra and
extracellular environments on the conformational equili-
brium of intrinsically disordered peptides. Applied to AD,
it shows the similarities and differences between conforma-
tional properties of Aβ peptides under conditions of intra-
cellular crowding and in non-polar environments (such as
imposed by the neuronal lipid membrane). Specifically,
both the crowding and the non-polar solvation make the
peptides less disordered, which may result in the increase
of their propensity for aggregation. Among the differences
are the opposite trends in bend and turn secondary struc-
ture contents under different conditions. We also show
that the 3D-RISM-KH based methodology could be used
to predict effects of synaptic metabolites and other small
molecules (e.g. therapeutic agents) on the structure of the
peptides. This feature of the methodology can be used to
screen potential inhibitors of pathological misfolding of
proteins in neurodegeneration.

37. *Spontaneous neurodegeneration in transgenic mice caused by histidine to tyrosine substitution at non-OR copper-binding site*

Alba Marín-Moreno<sup>a*</sup>, Thanh Hoa Tran<sup>b*</sup>, Juan Carlos Espinosa<sup>a</sup>, Edoardo Bistaffa<sup>c</sup>, Fabio Moda<sup>c</sup>, Juan María Torres<sup>a</sup> and Giuseppe Legname<sup>b</sup>

<footnote><sup>a</sup>University of Alberta, Department of Mechanical Engineering, Edmonton, Canada; <sup>b</sup>NRC Nanotechnology Research Centre, Edmonton, Canada; <sup>c</sup>University of Alberta, Departments of Biological Sciences and Computing Science, Edmonton, Canada; <sup>d</sup>University of British Columbia, Department of Medicine, Vancouver, Canada</footnote>
<figure><img src="image_26.png" /></figure>

<sup>a</sup>Centro de Investigación en Sanidad Animal (CISA-INIA), Madrid, Spain; <sup>b</sup>Laboratory of Prion Biology, Department of Neuroscience, Scuola Internazionale Superiore di Studi Avanzati, Trieste, Italy; <sup>c</sup>Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy

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

*These authors contributed equally to the work

## ABSTRACT

Prion protein has long been known as a copper binding protein via histidine residues in the octapeptide repeats (OR) and the non-OR region located in the disordered N-terminal tail of the protein. The functional implication of copper binding to PrP is not yet clear, but copper seems to play an important factor in prion disease.

Transgenic mice expressing the mouse prion protein replacing histidine (H) by tyrosine (Y) at codon 95 to disrupt the non-OR copper-binding site have been generated.

Mice overexpressing approximately two fold PrP H95Y showed clinical signs and died at about 100 days with spongiform degeneration and atypical PK-resistant PrP in the brain. Inoculation of brain homogenate from terminally ill mice overexpressing PrP H95Y to mice expressing a low level of PrP H95Y (1 fold) or Tga20 mice also causes lethal, spongiform encephalopathy.

H95Y substitution at non-OR region could promote PrP<sup>C</sup>-PrP<sup>Sc</sup> conversion and induce spontaneous neurodegeneration disease in transgenic mice, implying a critical role for the non-OR copper binding site in prion disease.

## KEYWORDS

Scrapie; prion strain; transmission barrier; mouse bioassay

## Funding

Spanish Ministerio de Ciencia, Innovación y Universidades (AGL2016-78,054R).

## 38. Signalling pathways underlying prion neurotoxicity

Nhat T. Le, Robert C. Mercer and David A. Harris

Department of Biochemistry, Boston University School of Medicine, Boston MA, United States of America

## ABSTRACT

The process by which prions propagate is now well understood; however, the molecular mechanism responsible for their neurotoxicity remains unclear. Synaptic loss is one of the earliest events in both *in vivo* and *in vitro* models of prion disease. Recently, we have developed sophisticated neuronal cell culture models to analyse the mechanisms

of prion-induced synaptic degeneration in a more physiologically relevant setting. Using this system to dissect the underlying cellular and molecular mechanisms, we show that exposure of hippocampal neurons to PrP<sup>Sc</sup> results in rapid, PrP<sup>C</sup>-dependent retraction of dendritic spines. In this process, PrP<sup>Sc</sup> activation of AMPA and NMDA receptors induces calcium influx, which subsequently activates p38 MAPK and its downstream MK2/3 kinases. Pharmacologically and genetically reducing p38 MAPK activity not only blocks PrP<sup>Sc</sup>-induced loss of dendritic spines, but also reverses dendritic spine loss that has already occurred. In order to identify additional components of prion-induced synaptotoxic pathways, we measured changes in the phosphoproteome and transcriptome of cultured neurons exposed to PrP<sup>Sc</sup> while they are undergoing well-defined alterations in synaptic structure and function. This work identifies a new, drugable target that could be used to block synaptotoxic sequelae in prion diseases: p38 MAPK. We are in the process of testing whether reducing p38 MAPK function, either genetically or pharmacologically, ameliorates the symptoms of prion disease in mice.

## 39. Generation of bona fide prions by large-scale Protein Misfolding Cyclic Amplification

Fei Wang, Luis Concha-Marambio, Enrique Armijo and Claudio Soto

Mitchell Center for Alzheimer's Disease and Related Brain Disorders, Department of Neurology, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, Texas, USA

**CONTACT** Fei Wang Fei.Wang.1@uth.tmc.edu

## ABSTRACT

The misfolding of normal prion protein (PrP<sup>C</sup>) into pathogenic PrP<sup>Sc</sup> is the key event causing prion diseases. However, the molecular mechanism underlying such conformational conversion remains elusive, largely due to the absence of high-resolution structural information of PrP<sup>Sc</sup>, hence impeding the developments of efficacious therapeutic strategies against these currently incurable diseases. To facilitate the structural studies of PrP<sup>Sc</sup>, here we present our latest results of large-scale production of bona fide prions by Protein Misfolding Cyclic Amplification (PMCA), which not only provides the platform for ultra-sensitive detection of prions in biological samples, but can also faithfully reproduce the key properties of prions, including infectivity, species barrier, and strain variability. In a pilot study, normal mouse brain homogenates were seeded with RML prion in large containers to produce bona fide prions by PMCA in reaction
<figure><img src="image_30.png" /></figure>

<sup>a</sup>Centro de Investigación en Sanidad Animal (CISA-INIA), Madrid, Spain; <sup>b</sup>Laboratory of Prion Biology, Department of Neuroscience, Scuola Internazionale Superiore di Studi Avanzati, Trieste, Italy; <sup>c</sup>Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy

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

*These authors contributed equally to the work

## ABSTRACT

Prion protein has long been known as a copper binding protein via histidine residues in the octapeptide repeats (OR) and the non-OR region located in the disordered N-terminal tail of the protein. The functional implication of copper binding to PrP is not yet clear, but copper seems to play an important factor in prion disease.

Transgenic mice expressing the mouse prion protein replacing histidine (H) by tyrosine (Y) at codon 95 to disrupt the non-OR copper-binding site have been generated.

Mice overexpressing approximately two fold PrP H95Y showed clinical signs and died at about 100 days with spongiform degeneration and atypical PK-resistant PrP in the brain. Inoculation of brain homogenate from terminally ill mice overexpressing PrP H95Y to mice expressing a low level of PrP H95Y (1 fold) or Tga20 mice also causes lethal, spongiform encephalopathy.

H95Y substitution at non-OR region could promote PrP<sup>C</sup>-PrP<sup>Sc</sup> conversion and induce spontaneous neurodegeneration disease in transgenic mice, implying a critical role for the non-OR copper binding site in prion disease.

## KEYWORDS

Scrapie; prion strain; transmission barrier; mouse bioassay

## Funding

Spanish Ministerio de Ciencia, Innovación y Universidades (AGL2016-78,054R).

## 38. Signalling pathways underlying prion neurotoxicity

Nhat T. Le, Robert C. Mercer and David A. Harris

Department of Biochemistry, Boston University School of Medicine, Boston MA, United States of America

## ABSTRACT

The process by which prions propagate is now well understood; however, the molecular mechanism responsible for their neurotoxicity remains unclear. Synaptic loss is one of the earliest events in both *in vivo* and *in vitro* models of prion disease. Recently, we have developed sophisticated neuronal cell culture models to analyse the mechanisms

of prion-induced synaptic degeneration in a more physiologically relevant setting. Using this system to dissect the underlying cellular and molecular mechanisms, we show that exposure of hippocampal neurons to PrP<sup>Sc</sup> results in rapid, PrP<sup>C</sup>-dependent retraction of dendritic spines. In this process, PrP<sup>Sc</sup> activation of AMPA and NMDA receptors induces calcium influx, which subsequently activates p38 MAPK and its downstream MK2/3 kinases. Pharmacologically and genetically reducing p38 MAPK activity not only blocks PrP<sup>Sc</sup>-induced loss of dendritic spines, but also reverses dendritic spine loss that has already occurred. In order to identify additional components of prion-induced synaptotoxic pathways, we measured changes in the phosphoproteome and transcriptome of cultured neurons exposed to PrP<sup>Sc</sup> while they are undergoing well-defined alterations in synaptic structure and function. This work identifies a new, drugable target that could be used to block synaptotoxic sequelae in prion diseases: p38 MAPK. We are in the process of testing whether reducing p38 MAPK function, either genetically or pharmacologically, ameliorates the symptoms of prion disease in mice.

## 39. Generation of bona fide prions by large-scale Protein Misfolding Cyclic Amplification

Fei Wang, Luis Concha-Marambio, Enrique Armijo and Claudio Soto

Mitchell Center for Alzheimer's Disease and Related Brain Disorders, Department of Neurology, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, Texas, USA

**CONTACT** Fei Wang Fei.Wang.1@uth.tmc.edu

## ABSTRACT

The misfolding of normal prion protein (PrP<sup>C</sup>) into pathogenic PrP<sup>Sc</sup> is the key event causing prion diseases. However, the molecular mechanism underlying such conformational conversion remains elusive, largely due to the absence of high-resolution structural information of PrP<sup>Sc</sup>, hence impeding the developments of efficacious therapeutic strategies against these currently incurable diseases. To facilitate the structural studies of PrP<sup>Sc</sup>, here we present our latest results of large-scale production of bona fide prions by Protein Misfolding Cyclic Amplification (PMCA), which not only provides the platform for ultra-sensitive detection of prions in biological samples, but can also faithfully reproduce the key properties of prions, including infectivity, species barrier, and strain variability. In a pilot study, normal mouse brain homogenates were seeded with RML prion in large containers to produce bona fide prions by PMCA in reaction
<figure><img src="image_27.png" /></figure>

volumes ranging from 30 mL to 120 mL. The prion yielded in this large-scale production maintained the RML prion strain characteristics in terms of infectivity in wild type animals, incubation time, $PrP^{Sc}$ biochemical profile and pathological changes. In the follow-up study, recombinant prion generated from bacterially expressed full-length recombinant mouse PrP seeded with RML prion was used to seed the large-scale production of recombinant $PrP^{Sc}$ by PMCA. The large-scale PMCA (LS-PMCA) platform is capable of producing at least 2 mg of PK-resistant recombinant $PrP^{Sc}$ in a 200-mL reaction in 24 h and has the option to scale up to as much as 1000 mL per round of LS-PMCA. The infectivity assessment of the recombinant $PrP^{Sc}$ from LS-PMCA is currently ongoing. Availability of a procedure to produce massive amounts of $PrP^{Sc}$ using recombinant protein as substrate which maintain the typical biological and biochemical properties of bona fide prions, will facilitate structural and biochemical studies of infectious prions.

## 40. Impact of different recombinant PrP substrates on the signalling response in the RT-QuIC

Susana Correia, Matthias Schmitz and Inga Zerr

Department of Neurology, Medical University of Goettingen, Goettingen, Germany

CONTACT Susana Correia Susana.correia@med.uni-goettingen.de

### ABSTRACT

Transmissible spongiform encephalopathies (TSE) are a group of neurodegenerative disorders, such as Creutzfeldt-Jakob disease (CJD), which are based on the conformational change of the prion protein (PrP) from the cellular form, PrPC, to the infectious scrapie form, PrPSc.

Based on the mechanism that PrPSc acts as a template for the conversion of PrPC, protein misfolding amplification assays, such as the real-time quaking-induced conversion (RT-QuIC) has been applied for the detection of misfolded PrPSc in the brain and cerebrospinal fluid (CSF) for the pre-mortem diagnosis of human prion diseases.

The technique bears a huge diagnostic and analytical potential. However the clinical variability of different sCJD subtypes (MM1, MM2, MV1, MV2, VV1, and VV2) has a relevant impact on the accuracy of diagnostic assays. An additional important factor is the kind of recombinant PrP substrate (e.g. hamster, hamster-sheep chimera, bank vole, etc.) used in the RT-QuIC. The diagnostic accuracy for different sCJD subtypes, familial forms – or the variant form of CJD is variable, e.g. only

a very few recombinant PrP substrates are able to detect variant CJD prions, such as bank vole PrP.

In our study, we aim to increase the diagnostic accuracy and the application spectrum of the RT-QuIC for all types of prion diseases, in particular for the atypical and rare forms like sCJD (MM2T, MM2C, and VV1), FFI or GSS. We will produce and purify a panel of different recombinant PrP substrates and compared the sensitivity and specificity among them across the wide spectrum of prion diseases to reach an optimized diagnostic accuracy.

## 41. A screen for prion and a -synuclein aggregate decontaminants to enhance safety of surgical instrument reprocessing

Daniel Heinzerª, Merve Avarª, Manuela Pfammatterª, Rita Moosª, Linda Irpinioª, Dezirae Schneiderª, Benjamin Kuhnᵇ, Stefan Mauerhoferᵇ, Urs Rosenbergᵇ, Adriano Aguzziª, Simone Hornemannª

CONTACT Simone Hornemann simone.hornemann@usz.ch

### ABSTRACT

Prions, the infectious agents causing prion diseases such as the Creutzfeldt-Jakob disease, show extreme robustness to common decontamination procedures. Therefore, there is a risk of iatrogenic transmission of these lethal diseases by inefficiently decontaminated medical instruments. So far, improvement of effective decontaminating agents applicable to routine sterilization procedures has not been feasible as the traditional assays for measuring prion infectivity are time-consuming and constrained by a limited throughput. The development of the real-time quaking induced conversion (RT-QuIC) assay [1], a fast and ultra-sensitive method, based on the self-propagation of proteinaceous agents in misfolding diseases, provides a new approach for the detection of infectious prions.

The adaption of this assay to a carrier mimicking surgical steel allows evaluating the efficiency of novel potential decontaminants in a fast and proficient way. In addition, with the emergence of evidence of α-synucleinopathies exhibiting prion-like infectious properties, this method was further adapted and applied to assess inactivation procedures for potentially hazardous α-synuclein fibrils. We demonstrated that the prion RT-QuIC assay is suitable to detect propagating prions adsorbed to 316L steel type metal beads, and that the treatment with the standard prion decontamination procedure (1 M NaOH for 2 h) completely abolished a positive signal in the assay. Around 120 different formulations were tested for their

<footnote>ªInstitute of Neuropathology, University of Zurich, Zurich, Switzerland; ᵇBorer Chemie AG, Zuchwil, Switzerland</footnote>
<figure><img src="image_31.png" /></figure>

volumes ranging from 30 mL to 120 mL. The prion yielded in this large-scale production maintained the RML prion strain characteristics in terms of infectivity in wild type animals, incubation time, $PrP^{Sc}$ biochemical profile and pathological changes. In the follow-up study, recombinant prion generated from bacterially expressed full-length recombinant mouse PrP seeded with RML prion was used to seed the large-scale production of recombinant $PrP^{Sc}$ by PMCA. The large-scale PMCA (LS-PMCA) platform is capable of producing at least 2 mg of PK-resistant recombinant $PrP^{Sc}$ in a 200-mL reaction in 24 h and has the option to scale up to as much as 1000 mL per round of LS-PMCA. The infectivity assessment of the recombinant $PrP^{Sc}$ from LS-PMCA is currently ongoing. Availability of a procedure to produce massive amounts of $PrP^{Sc}$ using recombinant protein as substrate which maintain the typical biological and biochemical properties of bona fide prions, will facilitate structural and biochemical studies of infectious prions.

## 40. Impact of different recombinant PrP substrates on the signalling response in the RT-QuIC

Susana Correia, Matthias Schmitz and Inga Zerr

Department of Neurology, Medical University of Goettingen, Goettingen, Germany

CONTACT Susana Correia Susana.correia@med.uni-goettingen.de

### ABSTRACT

Transmissible spongiform encephalopathies (TSE) are a group of neurodegenerative disorders, such as Creutzfeldt-Jakob disease (CJD), which are based on the conformational change of the prion protein (PrP) from the cellular form, PrPC, to the infectious scrapie form, PrPSc.

Based on the mechanism that PrPSc acts as a template for the conversion of PrPC, protein misfolding amplification assays, such as the real-time quaking-induced conversion (RT-QuIC) has been applied for the detection of misfolded PrPSc in the brain and cerebrospinal fluid (CSF) for the pre-mortem diagnosis of human prion diseases.

The technique bears a huge diagnostic and analytical potential. However the clinical variability of different sCJD subtypes (MM1, MM2, MV1, MV2, VV1, and VV2) has a relevant impact on the accuracy of diagnostic assays. An additional important factor is the kind of recombinant PrP substrate (e.g. hamster, hamster-sheep chimera, bank vole, etc.) used in the RT-QuIC. The diagnostic accuracy for different sCJD subtypes, familial forms – or the variant form of CJD is variable, e.g. only

a very few recombinant PrP substrates are able to detect variant CJD prions, such as bank vole PrP.

In our study, we aim to increase the diagnostic accuracy and the application spectrum of the RT-QuIC for all types of prion diseases, in particular for the atypical and rare forms like sCJD (MM2T, MM2C, and VV1), FFI or GSS. We will produce and purify a panel of different recombinant PrP substrates and compared the sensitivity and specificity among them across the wide spectrum of prion diseases to reach an optimized diagnostic accuracy.

## 41. A screen for prion and a -synuclein aggregate decontaminants to enhance safety of surgical instrument reprocessing

Daniel Heinzerª, Merve Avarª, Manuela Pfammatterª, Rita Moosª, Linda Irpinioª, Dezirae Schneiderª, Benjamin Kuhnᵇ, Stefan Mauerhoferᵇ, Urs Rosenbergᵇ, Adriano Aguzziª, Simone Hornemannª

ªInstitute of Neuropathology, University of Zurich, Zurich, Switzerland; ᵇBorer Chemie AG, Zuchwil, Switzerland

CONTACT Simone Hornemann simone.hornemann@usz.ch

### ABSTRACT

Prions, the infectious agents causing prion diseases such as the Creutzfeldt-Jakob disease, show extreme robustness to common decontamination procedures. Therefore, there is a risk of iatrogenic transmission of these lethal diseases by inefficiently decontaminated medical instruments. So far, improvement of effective decontaminating agents applicable to routine sterilization procedures has not been feasible as the traditional assays for measuring prion infectivity are time-consuming and constrained by a limited throughput. The development of the real-time quaking induced conversion (RT-QuIC) assay [1], a fast and ultra-sensitive method, based on the self-propagation of proteinaceous agents in misfolding diseases, provides a new approach for the detection of infectious prions.

The adaption of this assay to a carrier mimicking surgical steel allows evaluating the efficiency of novel potential decontaminants in a fast and proficient way. In addition, with the emergence of evidence of α-synucleinopathies exhibiting prion-like infectious properties, this method was further adapted and applied to assess inactivation procedures for potentially hazardous α-synuclein fibrils. We demonstrated that the prion RT-QuIC assay is suitable to detect propagating prions adsorbed to 316L steel type metal beads, and that the treatment with the standard prion decontamination procedure (1 M NaOH for 2 h) completely abolished a positive signal in the assay. Around 120 different formulations were tested for their
<figure><img src="image_28.png" /></figure>

efficiency to inactivate or remove prions from the steel beads. We identified one potential candidate formulation with good material compatibility that strongly and reliably reduced prion propagation activity in the RT-QuIC assay. The candidate formulation and two further alkaline cleaner are currently validated in a cell and mouse bioassay to confirm their decontamination properties. In addition, we developed an α-synuclein RT-QuIC assay that we used to test the inactivation efficiencies of known prion decontaminants on α-synuclein preformed fibrils. Interestingly, some of the commonly used prion decontamination procedures were less effective to deactivate the propagation efficiency of α-synuclein preformed fibrils. This implies that the resistance of α-synuclein fibrils might differ or even exceed the one of bona fide prions, and that other decontamination procedures need to be applied for the effective deactivation of α-synuclein fibrils.

## Reference

[1] Wilham JM, Orrú CD, Bessen RA, et al. Rapid endpoint quantitation of prion seeding activity with sensitivity comparable to bioassays. PLoS Pathog. 2 December 2010;6(12):e1001217.

## 42. Multiple sclerosis brain transmits pathology to humanized transgenic mice

Shigeki Tsutsuiª, Shoraf Dadakhujaevaª, Francisca Cavalcante Meloª, Geert Schenkᵇ, Roel Klaverᵇ, Hugo Tedfordª, Najwa E Kadiª, Karen Cumminsª, Gui Fang Guoª, Andrew V Caprarielloª, Julianne Proftª, Shyamosree Roychoudhuryª, Kyoichi Tsutsuiª, V. Wee Yongª, Jeffrey T Josephª, Gerald W. Zamponiª, Jeroen JG Geurtsᵇ and Peter K Stysª

## ABSTRACT

The fundamental aetiology of multiple sclerosis (MS) remains unknown. Two competing theories propose that on the one hand, MS is driven by a dysregulation of the peripheral immune system promoting inflammatory attacks on the CNS, while an alternative model posits that an initial primary degenerative process may secondarily trigger autoimmunity. Our hypothesis is that MS, like many neurodegenerative diseases, might be a protein misfolding disorder driven by accumulation of pathological cytotoxic aggregates that can spread through the CSF and CNS. One defining property of such diseases is the transmission of pathology to susceptible hosts.

We have previously shown that intracerebral inoculation (i.c.) of MS brain homogenates into human prion protein (PrP) over-expressor mice induced significant MS-like pathology, together with cognitive disability. We have expanded our dataset using i.c. transmission of MS pathologies in larger numbers of animals (53 mice from 10 primary or secondary-progressive MS patients, 65 mice from 9 control donors, 30 mice from 3 chronic encephalitis brains, and 10 mice from other neurological diseases, such as AD and Lewy body disease), using additional immunohistochemical injury markers, which further supports the underlying hypothesis. Passaging (inoculation of naïve mice with brain homogenate of MS-inoculated mice) also continued to transmit pathology. MS-injected mouse brains exhibited significant degeneration of myelin and axonal damage with glial and astroglial activation in the corpus callosum, leukocortical junction, thalamus and peri-ventricular region, assessed by SMI-94, QD9, SMI-32, Iba-1 and GFAP immunohistochemistry, whereas mice injected with control human brain did not develop pathology. Importantly, PrP-immunodepleted MS brain homogenates, blunted transmission of pathology, further supporting a possible role of pathological PrP. Formic acid-resistant prion protein aggregates were detected in both white and grey matter regions. Protease resistance, often seen in more conventional prionopathies, was not detected in either human MS brain or MS-inoculated mice.

**Conclusion:** Our results are consistent with the hypothesis that MS might be a primary degenerative disorder caused by accumulation and propagation of atypical pathogenic PrP, that can transmit pathology to humanized murine hosts. We speculate that these toxic pathological conformers could circulate in the CSF and spread to various brain regions in contact with CSF, as is characteristic of human MS lesion distribution. The resulting degeneration of myelin and release of antigenic debris could secondarily trigger an autoimmune inflammatory response in immune-predisposed hosts. Together, such a convolution of a primary prion-dependent degeneration, with secondary inflammation could explain the broad spectrum of human MS phenotypes.

## 43. Molecular characterization of prion diseases in Denmark: the national reference center experience from 2002 to 2018

Aušrinė Areškevičiūtėª, Helle Broholmª, Linea C. Melchiorª, Piero Parchiᵇ, Anna Bartoletti-Stellaᵇ, Eva L. Lundª

<footnote>ªUniversity of Calgary, Calgary, AB, Canada; ᵇVU University Medical Center, Amsterdam, Netherlands</footnote>
<figure><img src="image_32.png" /></figure>

efficiency to inactivate or remove prions from the steel beads. We identified one potential candidate formulation with good material compatibility that strongly and reliably reduced prion propagation activity in the RT-QuIC assay. The candidate formulation and two further alkaline cleaner are currently validated in a cell and mouse bioassay to confirm their decontamination properties. In addition, we developed an α-synuclein RT-QuIC assay that we used to test the inactivation efficiencies of known prion decontaminants on α-synuclein preformed fibrils. Interestingly, some of the commonly used prion decontamination procedures were less effective to deactivate the propagation efficiency of α-synuclein preformed fibrils. This implies that the resistance of α-synuclein fibrils might differ or even exceed the one of bona fide prions, and that other decontamination procedures need to be applied for the effective deactivation of α-synuclein fibrils.

## Reference

[1] Wilham JM, Orrú CD, Bessen RA, et al. Rapid endpoint quantitation of prion seeding activity with sensitivity comparable to bioassays. PLoS Pathog. 2 December 2010;6(12):e1001217.

## 42. Multiple sclerosis brain transmits pathology to humanized transgenic mice

Shigeki Tsutsuiª, Shoraf Dadakhujaevª, Francisca Cavalcante Meloª, Geert Schenkᵇ, Roel Klaverᵇ, Hugo Tedfordª, Najwa E Kadiª, Karen Cumminsª, Gui Fang Guoª, Andrew V Caprarielloª, Julianne Proftª, Shyamosree Roychoudhuryª, Kyoichi Tsutsuiª, V. Wee Yongª, Jeffrey T Josephª, Gerald W. Zamponiª, Jeroen JG Geurtsᵇ and Peter K Stysª

## ABSTRACT

The fundamental aetiology of multiple sclerosis (MS) remains unknown. Two competing theories propose that on the one hand, MS is driven by a dysregulation of the peripheral immune system promoting inflammatory attacks on the CNS, while an alternative model posits that an initial primary degenerative process may secondarily trigger autoimmunity. Our hypothesis is that MS, like many neurodegenerative diseases, might be a protein misfolding disorder driven by accumulation of pathological cytotoxic aggregates that can spread through the CSF and CNS. One defining property of such diseases is the transmission of pathology to susceptible hosts.

We have previously shown that intracerebral inoculation (i.c.) of MS brain homogenates into human prion protein (PrP) over-expressor mice induced significant MS-like pathology, together with cognitive disability. We have expanded our dataset using i.c. transmission of MS pathologies in larger numbers of animals (53 mice from 10 primary or secondary-progressive MS patients, 65 mice from 9 control donors, 30 mice from 3 chronic encephalitis brains, and 10 mice from other neurological diseases, such as AD and Lewy body disease), using additional immunohistochemical injury markers, which further supports the underlying hypothesis. Passaging (inoculation of naïve mice with brain homogenate of MS-inoculated mice) also continued to transmit pathology. MS-injected mouse brains exhibited significant degeneration of myelin and axonal damage with glial and astroglial activation in the corpus callosum, leukocortical junction, thalamus and peri-ventricular region, assessed by SMI-94, QD9, SMI-32, Iba-1 and GFAP immunohistochemistry, whereas mice injected with control human brain did not develop pathology. Importantly, PrP-immunodepleted MS brain homogenates, blunted transmission of pathology, further supporting a possible role of pathological PrP. Formic acid-resistant prion protein aggregates were detected in both white and grey matter regions. Protease resistance, often seen in more conventional prionopathies, was not detected in either human MS brain or MS-inoculated mice.

**Conclusion:** Our results are consistent with the hypothesis that MS might be a primary degenerative disorder caused by accumulation and propagation of atypical pathogenic PrP, that can transmit pathology to humanized murine hosts. We speculate that these toxic pathological conformers could circulate in the CSF and spread to various brain regions in contact with CSF, as is characteristic of human MS lesion distribution. The resulting degeneration of myelin and release of antigenic debris could secondarily trigger an autoimmune inflammatory response in immune-predisposed hosts. Together, such a convolution of a primary prion-dependent degeneration, with secondary inflammation could explain the broad spectrum of human MS phenotypes.

## 43. Molecular characterization of prion diseases in Denmark: the national reference center experience from 2002 to 2018

Aušrinė Areškevičiūtėª, Helle Broholmª, Linea C. Melchiorª, Piero Parchiᵇ, Anna Bartoletti-Stellaᵇ, Eva L. Lundª

<footnote>ªUniversity of Calgary, Calgary, AB, Canada; ᵇVU University Medical Center, Amsterdam, Netherlands</footnote>