<figure><img src="image_29.png" /></figure>

ªDanish Reference Center for Prion Diseases, Department of Pathology, Copenhagen University Hospital - Rigshospitalet, Copenhagen, Denmark; ᵇIRCCS Istituto delle Scienze Neurologiche di Bologna, Ospedale Bellaria, Bologna, Italy

CONTACT Aušrinė Areškevičiūtė ausrine.areskeviciute@regionh.dk

## ABSTRACT

We present a Danish cohort of prion disease cases with well-characterized fresh frozen brain samples that provides a great foundation for future research.

The aim of this study was an up-to-date molecular characterization and classification of all fresh frozen prion disease samples delivered to the Danish Reference Center for Prion Diseases in the period of 2002 and 2018.

Molecular characterization and classification of these samples was based on an improved immunoblotting method and sequencing of the whole prion protein gene coding region. Immuno- and histopathological findings in the brain tissues were re-evaluates in several cases. Available medical records were gathered for estimation of patients' age at disease onset and death, disease duration, and disease clinical presentation.

Thirty-seven of forty-one samples were classified as sporadic prion diseases with the following subtypes: MM/MV1 (n = 23), VV2 (n = 7), MM/MV1+2 (n = 3), MV2 (n = 2), MM2 (n = 2). A case of CJD with sporadic phenotype that occurred to a mother and wife of P102L mutation carriers was also a part of the Danish prion diseases cohort [1].

Four of forty-one cohort samples were found to harbor mutations in the prion protein gene. Two of those mutations were unique 5 and 8 octapeptide repeat insertions [2]. The other two mutations found in the cohort were a well-known P102L, and a novel T201S point mutations. The pathogenicity of T201S was recently investigated and described [3].

Re-evaluation of the cohort samples revealed that initial disease classification was inaccurate or missing in nearly every 5th case, which is a strong argument for consideration to re-evaluate older samples as techniques with higher sensitivity and specificity are being developed, and the classification system updated.

## References

[1] Areškevičiūtė, et al. JNEN 2018;77:673-684.

[2] Areškevičiūtė, et al. A novel eight octapeptide repeat insertion in PRNP causing prion disease in a Danish family. JNEN;2019:In Press.

[3] Mok, et al. Neurobiol Aging. 2018;71:265.e1-265.e7.

# 44. Understanding prion-like mechanisms in traumatic brain injury using a novel in vivo model

Hadeel Alyenbaawiª,ᵇ and W. Ted Allisonª,ᵇ,ᶜ

ªCenter for Prion & Protein Folding Diseases, University of Alberta, Edmonton AB, Canada; ᵇDepartment of Medical Genetics, University of Alberta, Edmonton AB, Canada; ᶜDepartment of Biological Sciences, University of Alberta, Edmonton AB, Canada

CONTACT Hadeel Alyenbaawi alyenbaa@ualberta.ca

## ABSTRACT

**Background/Introduction:** Traumatic brain injury (TBI) is known as one of the risk factors leading to neurodegeneration and dementia, in particular chronic traumatic encephalopathy (CTE). CTE is one of the tauopathies characterized by a distinct accumulation of hyperphosphorylated tau. Similar to Alzheimer Disease (AD) and other tauopathies, pathology seems to progress to other regions during later stages of the disease, mimicking the spreading mechanisms of prion disease. The prion-like seeding and transmission of tau in CTE and TBI cases has begun to be illuminated by a limited number of *in vitro* and *in vivo* studies. However, there is still a lack of information regarding the mechanisms of prion-like spreading and cell uptake of tau seeds when it comes to TBI. We aimed to establish a novel TBI model using our novel transparent tau biosensor zebrafish that we engineered to detect and visualize tau seeds. There is much promise in establishing *in vivo* models to gain greater understanding of the mechanisms controlling the spreading of tau pathology in TBI patients.

**Material and Methods:** We successfully engineered a stable transgenic zebrafish line that expresses a tauopathy biosensor reporter protein that detects various forms of tau from two to four days post-transduction. We also optimized a simplified system to induce blast injury on the tauopathy biosensor zebrafish. Zebrafish subjected to blast injury were analysed for the formation of GFP+ puncta indicative of tau seeds in the brains and spinal cord at various time points. Additionally, we evaluated the impact of administering convulsant drugs as stress agents on the spreading and cell uptake of tau inclusions in both a dose- and time-dependent manner.

**Results:** The zebrafish subjected to blast injury exhibited seizure-like movement immediately following the injury. We could reliably detect GFP+ tau inclusions three to four days after subjecting the larvae to blast injury. The number of these tau inclusions was significantly higher in the blast injury group compared to the control group. Treatment with various concentrations
<figure><img src="image_33.png" /></figure>

<sup>a</sup>Danish Reference Center for Prion Diseases, Department of Pathology, Copenhagen University Hospital - Rigshospitalet, Copenhagen, Denmark; <sup>b</sup>IRCCS Istituto delle Scienze Neurologiche di Bologna, Ospedale Bellaria, Bologna, Italy

CONTACT Aušrinė Areškevičiūtė ausrine.areskeviciute@regionh.dk

## ABSTRACT

We present a Danish cohort of prion disease cases with well-characterized fresh frozen brain samples that provides a great foundation for future research.

The aim of this study was an up-to-date molecular characterization and classification of all fresh frozen prion disease samples delivered to the Danish Reference Center for Prion Diseases in the period of 2002 and 2018.

Molecular characterization and classification of these samples was based on an improved immunoblotting method and sequencing of the whole prion protein gene coding region. Immuno- and histopathological findings in the brain tissues were re-evaluates in several cases. Available medical records were gathered for estimation of patients' age at disease onset and death, disease duration, and disease clinical presentation.

Thirty-seven of forty-one samples were classified as sporadic prion diseases with the following subtypes: MM/MV1 (n = 23), VV2 (n = 7), MM/MV1+2 (n = 3), MV2 (n = 2), MM2 (n = 2). A case of CJD with sporadic phenotype that occurred to a mother and wife of P102L mutation carriers was also a part of the Danish prion diseases cohort [1].

Four of forty-one cohort samples were found to harbor mutations in the prion protein gene. Two of those mutations were unique 5 and 8 octapeptide repeat insertions [2]. The other two mutations found in the cohort were a well-known P102L, and a novel T201S point mutations. The pathogenicity of T201S was recently investigated and described [3].

Re-evaluation of the cohort samples revealed that initial disease classification was inaccurate or missing in nearly every 5th case, which is a strong argument for consideration to re-evaluate older samples as techniques with higher sensitivity and specificity are being developed, and the classification system updated.

## References

[1] Areškevičiūtė, et al. JNEN 2018;77:673-684.

[2] Areškevičiūtė, et al. A novel eight octapeptide repeat insertion in PRNP causing prion disease in a Danish family. JNEN;2019:In Press.

[3] Mok, et al. Neurobiol Aging. 2018;71:265.e1-265.e7.

# 44. Understanding prion-like mechanisms in traumatic brain injury using a novel in vivo model

Hadeel Alyenbaawi<sup>a,b</sup> and W. Ted Allison<sup>a,b,c</sup>

<sup>a</sup>Center for Prion & Protein Folding Diseases, University of Alberta, Edmonton AB, Canada; <sup>b</sup>Department of Medical Genetics, University of Alberta, Edmonton AB, Canada; <sup>c</sup>Department of Biological Sciences, University of Alberta, Edmonton AB, Canada

CONTACT Hadeel Alyenbaawi alyenbaa@ualberta.ca

## ABSTRACT

**Background/Introduction:** Traumatic brain injury (TBI) is known as one of the risk factors leading to neurodegeneration and dementia, in particular chronic traumatic encephalopathy (CTE). CTE is one of the tauopathies characterized by a distinct accumulation of hyperphosphorylated tau. Similar to Alzheimer Disease (AD) and other tauopathies, pathology seems to progress to other regions during later stages of the disease, mimicking the spreading mechanisms of prion disease. The prion-like seeding and transmission of tau in CTE and TBI cases has begun to be illuminated by a limited number of *in vitro* and *in vivo* studies. However, there is still a lack of information regarding the mechanisms of prion-like spreading and cell uptake of tau seeds when it comes to TBI. We aimed to establish a novel TBI model using our novel transparent tau biosensor zebrafish that we engineered to detect and visualize tau seeds. There is much promise in establishing *in vivo* models to gain greater understanding of the mechanisms controlling the spreading of tau pathology in TBI patients.

**Material and Methods:** We successfully engineered a stable transgenic zebrafish line that expresses a tauopathy biosensor reporter protein that detects various forms of tau from two to four days post-transduction. We also optimized a simplified system to induce blast injury on the tauopathy biosensor zebrafish. Zebrafish subjected to blast injury were analysed for the formation of GFP+ puncta indicative of tau seeds in the brains and spinal cord at various time points. Additionally, we evaluated the impact of administering convulsant drugs as stress agents on the spreading and cell uptake of tau inclusions in both a dose- and time-dependent manner.

**Results:** The zebrafish subjected to blast injury exhibited seizure-like movement immediately following the injury. We could reliably detect GFP+ tau inclusions three to four days after subjecting the larvae to blast injury. The number of these tau inclusions was significantly higher in the blast injury group compared to the control group. Treatment with various concentrations
<figure><img src="image_30.png" /></figure>

of convulsant drugs affected the formation of those
inclusions in a dose-dependent manner.

**Conclusions:** The engineering of an *in vivo* tau biosensor and TBI model in larvae zebrafish will uncover information surrounding prion-like mechanisms of tau pathology and will also serve as a valuable model for drug screening and intervention.

**45. Two prion protein paralogs of zebrafish have opposing impacts on seizure susceptibility**

Laszlo F. Locskai, Richard Kanyo, Patricia L.A. Leighton and W. Ted Allison

Centre for Prions & Protein Folding Disease, University of Alberta,
Edmonton AB, Canada; Department of Biological Sciences,
University of Alberta, Edmonton AB, Canada

ABSTRACT

**Background:** Cellular prion protein (PrP<sup>C</sup>) is famous from its misfolded counterpart, PrP<sup>Sc</sup>, which is involved in neurodegenerative diseases such as Creutzfeldt Jakob's disease. PrP<sup>C</sup> has also been linked to Alzheimer's disease, the most prevalent form of dementia, by direct interactions with amyloid-β oligomers and amyloid-β precursor protein (APP). Our focus here is on understanding PrP and APP physiological interactions and functions. Reduced protein functions likely impact disease progression, hampering the engineering of potential therapeutics. Using the mutant zebrafish we have engineered, we seek to explore the mechanism of neural regulation attributable to zebrafish homologs of PrP<sup>C</sup>.

**Materials:** We have engineered mutant zebrafish with disruptions in homologs of *PRNP* (encoding PrP<sup>C</sup>) and *APP*, being *prp1<sup>-/-</sup>*, *prp2<sup>-/-</sup>* or compound *prp1<sup>-/-</sup>*, *prp2<sup>-/-</sup>*, and *appa<sup>-/-</sup>*. Zebrafish larvae were treated with convulsants pentylenetetrazol (PTZ) or kainic acid (KA) via bath treatment to induce seizures. Seizure responses were measured by EthoVision behavioural tracking software. The neural activity of free-swimming larvae was also quantified during induced seizures by genetically encoded Calcium Modulated Photoactivatable Ratiometric Integrator (CaMPARI).

**Results:** When induced with the convulsant PTZ, *prp2<sup>-/-</sup>* mutants have a large increase in seizure response compared to wildtype whereas *prp1<sup>-/-</sup>* exhibit a more modest increase. Neural activity in the hindbrain measured by CaMPARI presented parallel findings insomuch that PTZ induced increased activity in *prp2<sup>-/-</sup>* zebrafish compared to wildtype. Initial results with KA suggest zebrafish exhibit a decrease in locomotor activity in *prp1* mutants compared to wildtype. This contrasts compound mutant disruption, which appears to be similar

to locomotor activity versus wildtype. PTZ induction of *appa<sup>-/-</sup>* larvae caused an increased seizure response of a similar PTZ dose-response profile, but higher in magnitude to *prp1<sup>-/-</sup>*.

**Conclusions:** These results suggest that PrP<sup>C</sup> homologs have taken on non-additive or perhaps opposing functions in zebrafish neurophysiology, and that *appa*<sup>-/-</sup> functions in neuroprotection. It appears each paralog may regulate ion channels in opposing ways to mediate their neuroprotective function, though further research is warranted. Understanding which specific channels are regulated by each protein paralog, and in what fashion, has potential to illuminate the mechanism of PrP<sup>C</sup> and APP function at the synapse.

**46. Characterization of Syrian hamster adapted H-BSE prion**

Kohtaro Miyazawa<sup>a</sup>, Kentaro Masujin<sup>b</sup>, Yuichi Matsuura<sup>a</sup>, Yoshifumi Iwamaru<sup>a</sup>

<sup>a</sup>Prion disease unit, National Institute of Animal Health (NIAH), National Agriculture and Food Research Organization (NARO), Tsukuba, Ibaraki, Japan; <sup>b</sup>Exotic Disease Research Unit, Division of Transboundary Animal Diseases, NIAH, NARO, Kodaira, Tokyo, Japan

CONTACT Kohtaro Miyazawa miyazawak@affrc.go.jp

ABSTRACT

**Introduction:** Bovine spongiform encephalopathy (BSE) is a prion disease in cattle, which is characterized by spongiform changes and accumulation of a disease-associated isoform of prion protein (PrP<sup>d</sup>). Nowadays, BSE is classified into three types (classical type: C-BSE, high-type: H-BSE, and low-type: L-BSE) according to the electrophoretic migration of their proteinase K resistant core of PrP<sup>d</sup> (PrPres). Although Syrian hamster models have already proved to be useful for studying prion diseases, transmission of C- and H-BSE prions from cattle to Syrian hamsters has been inefficient. We previously reported that mouse-passaged C-BSE prion was transmissible to Syrian hamsters. Recently, we also achieved successful transmission of H-BSE prion from cattle to transgenic mice overexpressing hamster PrP (TgHaNSE). The distribution of immunola-beled PrP<sup>d</sup> in the brain was different between TgHaNSE mice infected with cattle H-BSE prion and those infected with mouse-passaged C-BSE prion even after the third passage in TgHaNSE mice. However, the molecular mass of the unglycosylated PrPres and glycoform profiles were indistinguishable between them. Because these results were obtained under artificial condition in TgHaNSE mice, we performed a subsequent transmission of TgHaNSE mouse-passaged H-BSE prion to Syrian hamsters and compared the biological and biochemical properties of Syrian
<figure><img src="image_34.png" /></figure>

of convulsant drugs affected the formation of those
inclusions in a dose-dependent manner.

**Conclusions:** The engineering of an *in vivo* tau biosensor and TBI model in larvae zebrafish will uncover information surrounding prion-like mechanisms of tau pathology and will also serve as a valuable model for drug screening and intervention.

**45. Two prion protein paralogs of zebrafish have opposing impacts on seizure susceptibility**

Laszlo F. Locskai, Richard Kanyo, Patricia L.A. Leighton and W. Ted Allison

Centre for Prions & Protein Folding Disease, University of Alberta,
Edmonton AB, Canada; Department of Biological Sciences,
University of Alberta, Edmonton AB, Canada

ABSTRACT

**Background:** Cellular prion protein (PrP<sup>C</sup>) is famous from its misfolded counterpart, PrP<sup>Sc</sup>, which is involved in neurodegenerative diseases such as Creutzfeldt Jakob's disease. PrP<sup>C</sup> has also been linked to Alzheimer's disease, the most prevalent form of dementia, by direct interactions with amyloid-β oligomers and amyloid-β precursor protein (APP). Our focus here is on understanding PrP and APP physiological interactions and functions. Reduced protein functions likely impact disease progression, hampering the engineering of potential therapeutics. Using the mutant zebrafish we have engineered, we seek to explore the mechanism of neural regulation attributable to zebrafish homologs of PrP<sup>C</sup>.

**Materials:** We have engineered mutant zebrafish with disruptions in homologs of *PRNP* (encoding PrP<sup>C</sup>) and *APP*, being *prp1<sup>-/-</sup>*, *prp2<sup>-/-</sup>* or compound *prp1<sup>-/-</sup>*, *prp2<sup>-/-</sup>*, and *appa<sup>-/-</sup>*. Zebrafish larvae were treated with convulsants pentylenetetrazol (PTZ) or kainic acid (KA) via bath treatment to induce seizures. Seizure responses were measured by EthoVision behavioural tracking software. The neural activity of free-swimming larvae was also quantified during induced seizures by genetically encoded Calcium Modulated Photoactivatable Ratiometric Integrator (CaMPARI).

**Results:** When induced with the convulsant PTZ, *prp2<sup>-/-</sup>* mutants have a large increase in seizure response compared to wildtype whereas *prp1<sup>-/-</sup>* exhibit a more modest increase. Neural activity in the hindbrain measured by CaMPARI presented parallel findings insomuch that PTZ induced increased activity in *prp2<sup>-/-</sup>* zebrafish compared to wildtype. Initial results with KA suggest zebrafish exhibit a decrease in locomotor activity in *prp1* mutants compared to wildtype. This contrasts compound mutant disruption, which appears to be similar

to locomotor activity versus wildtype. PTZ induction of *appa<sup>-/-</sup>* larvae caused an increased seizure response of a similar PTZ dose-response profile, but higher in magnitude to *prp1<sup>-/-</sup>*.

**Conclusions:** These results suggest that PrP<sup>C</sup> homologs have taken on non-additive or perhaps opposing functions in zebrafish neurophysiology, and that *appa*<sup>-/-</sup> functions in neuroprotection. It appears each paralog may regulate ion channels in opposing ways to mediate their neuroprotective function, though further research is warranted. Understanding which specific channels are regulated by each protein paralog, and in what fashion, has potential to illuminate the mechanism of PrP<sup>C</sup> and APP function at the synapse.

**46. Characterization of Syrian hamster adapted H-BSE prion**

Kohtaro Miyazawa<sup>a</sup>, Kentaro Masujin<sup>b</sup>, Yuichi Matsuura<sup>a</sup>, Yoshifumi Iwamaru<sup>a</sup>

<sup>a</sup>Prion disease unit, National Institute of Animal Health (NIAH), National Agriculture and Food Research Organization (NARO), Tsukuba, Ibaraki, Japan; <sup>b</sup>Exotic Disease Research Unit, Division of Transboundary Animal Diseases, NIAH, NARO, Kodaira, Tokyo, Japan

CONTACT Kohtaro Miyazawa miyazawak@affrc.go.jp

ABSTRACT

**Introduction:** Bovine spongiform encephalopathy (BSE) is a prion disease in cattle, which is characterized by spongiform changes and accumulation of a disease-associated isoform of prion protein (PrP<sup>d</sup>). Nowadays, BSE is classified into three types (classical type: C-BSE, high-type: H-BSE, and low-type: L-BSE) according to the electrophoretic migration of their proteinase K resistant core of PrP<sup>d</sup> (PrPres). Although Syrian hamster models have already proved to be useful for studying prion diseases, transmission of C- and H-BSE prions from cattle to Syrian hamsters has been inefficient. We previously reported that mouse-passaged C-BSE prion was transmissible to Syrian hamsters. Recently, we also achieved successful transmission of H-BSE prion from cattle to transgenic mice overexpressing hamster PrP (TgHaNSE). The distribution of immunola-beled PrP<sup>d</sup> in the brain was different between TgHaNSE mice infected with cattle H-BSE prion and those infected with mouse-passaged C-BSE prion even after the third passage in TgHaNSE mice. However, the molecular mass of the unglycosylated PrPres and glycoform profiles were indistinguishable between them. Because these results were obtained under artificial condition in TgHaNSE mice, we performed a subsequent transmission of TgHaNSE mouse-passaged H-BSE prion to Syrian hamsters and compared the biological and biochemical properties of Syrian
<figure><img src="image_31.png" /></figure>

hamster-adapted H-BSE prion to those of Syrian hamster-adapted C-BSE and L-BSE prions.

**Results:** Syrian hamsters inoculated with 10% brain homogenates of H-BSE prion-infected TgHaNSE mouse developed the neurological signs of the disease after long incubation periods of approximately 450 days. Even after the second passage, hamsters survived for ~390 days. The PrPres banding patterns and the glycoform profiles observed in H-BSE prion-infected TgHaNSE mice were maintained after the transmission into Syrian hamsters. Moreover, PrPres banding patterns of hamsters infected with H-BSE prion were identical with those of hamsters infected with C-BSE prion even after the second passage. On the other hand, the molecular mass of the unglycosylated PrPres of hamsters infected with L-BSE prion was slightly lower than those of hamsters infected with H- and C-BSE prions. Glycoform profiles were quite similar between hamsters infected with three different BSE prions. But the distribution of immunolabeled $Pr^d$ in the brain and the lesion profiles were different between them.

**Conclusion:** Our data suggest that the PrPres banding patterns and the glycoform profiles may be influenced by the properties of host $Pr^C$ rather than by prion strain. On the other hand, biological and pathological features including survival periods, $Pr^d$ distribution in the brain and the lesion profiles may be determined by prion strain itself. It is interesting that the ratio of di-glycosylated PrPres is dominant in Syrian hamsters infected with all three BSE prions. These models might be useful for investigations of the mechanisms underlying the conversion from $Pr^C$ to $Pr^Sc$.

**47. Sugar analogues and their mechanism of action as prion disease therapy**

Garrett J.B. Molnerª, Manjeet Kumarª,ᵇ and Valerie L. Simª,ᵇ

ªCentre for Prions and Protein Folding Diseases, University of Alberta; ᵇDepartment of Medicine, Division of Neurology, University of Alberta

**CONTACT** Garrett J.B. Molner gmolner@ualberta.ca

**ABSTRACT**

A central hallmark of prion disease is the conversion of normal cellular prion protein (PrPC) to a pathogenic, protease resistant form (PrPSc) that ultimately leads to neurodegeneration. Species devoid of prion protein are incapable of propagating prions and therefore are incapable of prion infection.¹ Interestingly, pathogenic prions are hypothesized to exist in strains which may induce distinct strain-specific pathologies and phenotypes. One

phenotypic strain characteristic is the relative amounts of un, mono, and di-glycosylated PrP.² While strain information is largely thought to be encoded in the protein core conformation, there is evidence to suggest a role for glycosylation in PrP conversion and trafficking.³⁻⁵ From this concept, both a potential therapeutic strategy and an opportunity to examine a role for glycosylation in strain-restricted conversion is possible.

We manipulated PrPC glycosylation in cell culture using naturally occurring sugar analogues and their alky-derivatives that target α- and β-glucosidases. Treatment led to the elimination of PrPSc in CAD5 cells chronically infected with RML strain prions. However, there were concomitant changes in PrPC levels, leading us to question whether the mechanism of action was indeed by alteration of conversion susceptibility of PrPC (with modified sugars), or whether this treatment led to a sequestration or degradation of PrPC, reducing its likelihood of conversion. To fully understand the mechanism of inhibition, we are using immunoblot, glycoform profiling, and confocal microscopy to determine the effect these compounds have on PrP glycosylation and cellular localization. Results will be discussed in the context of using sugar analogues as a prion therapeutic.

**References**

[1] Büeler H, et al. Cell 1993;73(7):1339-47.

[2] Khalili-Shirazi A, et al. J Gen Virol. 2005;86(9):2635-44.

[3] Makarava N, et al. Acta Neuropathol Com. 2018;6(1):92. 4.

[4] Li J, et al. Science. 2010;327(5967):869-72.

[5] Katorcha E, et al. Sci Rep. 2015;5:16,912.

**48. Transcriptomic analysis reveals conserved cross species functions of the cellular prion protein**

Niall M Pollockª,ᵇ, Patricia L. A Leightonª,ᵇ, Gavin J. Neilª and W. Ted Allisonª,ᵇ

ªDepartment of Biological Sciences, University of Alberta, Edmonton AB, Canada; ᵇCentre for Prion and Protein Folding Diseases, University of Alberta, Edmonton AB, Canada

**CONTACT** Niall M Pollock nmpolloc@ualberta.ca

**ABSTRACT**

**Introduction:** Cellular Prion Protein (PrP<sup>C</sup>) is well-studied due to being able to misfold into Scrapie Prion Protein (PrP<sup>SC</sup>) causing progressive and fatal neurodegenerative diseases both in humans and other animals. Normally folded PrP<sup>C</sup> is implicated in the pathology of Alzheimer's disease, the leading cause of dementia worldwide, through high affinity binding with soluble amyloid-β oligomers.
<figure><img src="image_35.png" /></figure>

hamster-adapted H-BSE prion to those of Syrian hamster-adapted C-BSE and L-BSE prions.

**Results:** Syrian hamsters inoculated with 10% brain homogenates of H-BSE prion-infected TgHaNSE mouse developed the neurological signs of the disease after long incubation periods of approximately 450 days. Even after the second passage, hamsters survived for ~390 days. The PrPres banding patterns and the glycoform profiles observed in H-BSE prion-infected TgHaNSE mice were maintained after the transmission into Syrian hamsters. Moreover, PrPres banding patterns of hamsters infected with H-BSE prion were identical with those of hamsters infected with C-BSE prion even after the second passage. On the other hand, the molecular mass of the unglycosylated PrPres of hamsters infected with L-BSE prion was slightly lower than those of hamsters infected with H- and C-BSE prions. Glycoform profiles were quite similar between hamsters infected with three different BSE prions. But the distribution of immunolabeled PrP<sup>d</sup> in the brain and the lesion profiles were different between them.

**Conclusion:** Our data suggest that the PrPres banding patterns and the glycoform profiles may be influenced by the properties of host PrP<sup>C</sup> rather than by prion strain. On the other hand, biological and pathological features including survival periods, PrP<sup>d</sup> distribution in the brain and the lesion profiles may be determined by prion strain itself. It is interesting that the ratio of di-glycosylated PrPres is dominant in Syrian hamsters infected with all three BSE prions. These models might be useful for investigations of the mechanisms underlying the conversion from PrP<sup>C</sup> to PrP<sup>Sc</sup>.

**47. Sugar analogues and their mechanism of action as prion disease therapy**

Garrett J.B. Molner<sup>a</sup>, Manjeet Kumar<sup>a,b</sup> and Valerie L. Sim<sup>a,b</sup>

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

**CONTACT** Garrett J.B. Molner gmolner@ualberta.ca

**ABSTRACT**

A central hallmark of prion disease is the conversion of normal cellular prion protein (PrPC) to a pathogenic, protease resistant form (PrPSc) that ultimately leads to neurodegeneration. Species devoid of prion protein are incapable of propagating prions and therefore are incapable of prion infection.1 Interestingly, pathogenic prions are hypothesized to exist in strains which may induce distinct strain-specific pathologies and phenotypes. One

phenotypic strain characteristic is the relative amounts of un, mono, and di-glycosylated PrP<sup>C</sup>. While strain information is largely thought to be encoded in the protein core conformation, there is evidence to suggest a role for glycosylation in PrP conversion and trafficking.3–5 From this concept, both a potential therapeutic strategy and an opportunity to examine a role for glycosylation in strain-restricted conversion is possible.

We manipulated PrPC glycosylation in cell culture using naturally occurring sugar analogues and their alky-derivatives that target α- and β-glucosidases. Treatment led to the elimination of PrPSc in CAD5 cells chronically infected with RML strain prions. However, there were concomitant changes in PrPC levels, leading us to question whether the mechanism of action was indeed by alteration of conversion susceptibility of PrPC (with modified sugars), or whether this treatment led to a sequestration or degradation of PrPC, reducing its likelihood of conversion. To fully understand the mechanism of inhibition, we are using immunoblot, glycoform profiling, and confocal microscopy to determine the effect these compounds have on PrP glycosylation and cellular localization. Results will be discussed in the context of using sugar analogues as a prion therapeutic.

**References**

[1] Büeler H, et al. Cell 1993;73(7):1339–47.

[2] Khalili-Shirazi A, et al. J Gen Virol. 2005;86(9):2635–44.

[3] Makarava N, et al. Acta Neuropathol Com. 2018;6(1):92. 4.

[4] Li J, et al. Science. 2010;327(5967):869–72.

[5] Katorcha E, et al. Sci Rep. 2015;5:16,912.

**48. Transcriptomic analysis reveals conserved cross species functions of the cellular prion protein**

Niall M Pollock<sup>a,b</sup>, Patricia L. A Leighton<sup>a,b</sup>, Gavin J. Neil<sup>a</sup> and W. Ted Allison<sup>a,b</sup>

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

**CONTACT** Niall M Pollock nmpolloc@ualberta.ca

**ABSTRACT**

**Introduction:** Cellular Prion Protein (PrP<sup>C</sup>) is well-studied due to being able to misfold into Scrapie Prion Protein (PrP<sup>SC</sup>) causing progressive and fatal neurodegenerative diseases both in humans and other animals. Normally folded PrP<sup>C</sup> is implicated in the pathology of Alzheimer's disease, the leading cause of dementia worldwide, through high affinity binding with soluble amyloid-β oligomers.
<figure><img src="image_32.png" /></figure>

However, the normal function(s) of PrPC are not well understood. We have used our *prp1*; *prp2* compound homozygous knockout mutant zebrafish to identify potentially conserved, cross-species functions of PrPC using RNA-sequencing analysis in addition to exploring the increasing evidence for PrPC involvement in cell adhesion.

**Methods:** Three-day post fertilization wild-type and *prp1*<sup>ua5003/ua5003</sup>; *prp2*<sup>ua5001/ua5001</sup> zebrafish larvae underwent PE100-125 HiDeq2500 RNA-sequencing. Three biological replicates each consisting of 50 pooled larvae were used for each genotype. Analysis of sequencing results was processed using Geneious R9/10 and R packages cufflinks and cummeRbund. Alterations in transcript abundance were confirmed using RT-qPCR for selected genes of interest. Further cell adhesion studies were carried out through *in situ* hybridization analysis and immunohistochemistry.

**Results:** RNA-sequencing analysis of mutant fish show large changes in transcript abundance (log₂ fold change of 0.5 or greater) of 1,249 genes compared to wild-type fish. Of interest are genes involved in cell adhesion, including *st8sia2*, *ncam1a* and members of the protocadherin family, which are thought to be involved during early development and continued maintenance of the central nervous system (CNS). Through search and comparison with published transcriptomes from embryonic PrPC knockout mice, we have identified many common affected biological processes in our mutant zebrafish including apoptosis, proteolysis, oxidative stress and cell proliferation, and adhesion. Further analysis on cell adhesion through RT-qPCR of *ncam1a* and *st8sia2* shows a significant reduction in transcript abundance between wild-type and mutant fish, with *in situ* hybridization of *ncam1a* confirming a decreased distribution of mRNA across the CNS.

**Conclusions:** Our transcriptomic sequencing analysis supports a role for PrPC during early development and sustainment of the CNS, seen through significant changes in transcript abundance in members of the protocadherin family and other genes responsible for cell adhesion and proliferation. We propose cell adhesion is an evolutionary conserved function of PrPC which will be of important consideration regarding how PrPC function may be affected during disease.

**KEYWORDS** Cellular prion protein; cell adhesion; RNA-sequencing; zebrafish

**49. The agent of transmissible mink encephalopathy passed in sheep is similar to BSE-L**

E. D. Cassmann<sup>a,b</sup>, S. J. Moore<sup>a,b</sup>, R. D. Kokemuller<sup>a</sup>, A. Balkema-Buschmann<sup>c</sup>, M. H. Groschup<sup>c</sup> and J. J. Greenlee<sup>a</sup>

<sup>a</sup>Virus and Prion Research Unit, National Animal Disease Center, ARS, United States Department of Agriculture, Ames, IA, USA (EDC, SJM, RDK, JJG); <sup>b</sup>Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the U.S. Department of Energy (DOE) and the U.S. Department of Agriculture (USDA). ORISE is managed by ORAU under DOE contract number DE-SC0014664. (EDC, SJM), Department of Veterinary Pathology, Iowa State University, Ames, IA, USA (JDS); <sup>c</sup>Institute of Novel and Emerging Infectious Diseases, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Greifswald - Isle of Riems, Germany (ABB, MHG)

**CONTACT** E. D. Cassmann <eric.cassmann@usda.gov>

**ABSTRACT**

**Introduction:** Transmissible mink encephalopathy (TME) is a fatal neurologic prion disease of farmed mink. Epidemiologic and experimental evidence following a Wisconsin outbreak in 1985 has linked TME to low-type bovine spongiform encephalopathy (BSE-L). Evidence suggests that farmed mink were likely exposed through feeding of BSE-L infected downer cattle. The interspecies transmission of TME to cattle has been documented. Recently, we demonstrated the susceptibility of sheep to cattle passaged TME by intracranial inoculation. The aim of the present study was to compare ovine passaged cattle TME to other prion diseases of food-producing animals. Using a bovine transgenic mouse model, we compared the disease phenotype of sheep TME to BSE-C and BSE-L.

**Materials and Methods:** Separate inoculants of sheep passaged TME were derived from animals with the VRQ/VRQ (VV<sub>136</sub>) and ARQ/VRQ (AV<sub>136</sub>) prion protein genotype. Transgenic bovinized mice (TgBovXV) were intracranially inoculated with 20 µl of 1% w/v brain homogenate. The disease phenotypes were characterized by comparing the attack rates, incubation periods, and vacuolation profiles in TgBovXV mice.

**Results:** The attack rate for BSE-C (13/13), BSE-L (18/18), and TME<sub>VV</sub> (21/21) was 100%; whereas, the TME<sub>AV</sub> group (15/19) had an incomplete attack rate. The average incubation periods were 299, 280, 310, and 541 days, respectively. The vacuolation profiles of BSE-L and TME<sub>VV</sub> were most similar with mild differences observed in the thalamus and medulla. Vacuolation profiles from the BSE-C and TME<sub>AV</sub> experimental groups were different than TME<sub>VV</sub> and BSE-L.

**Conclusion:** Overall the phenotype of disease in TME inoculated transgenic mice was dependent on the sheep donor genotype (VV vs AV). The results of the present study indicate that TME isolated from VRQ/VRQ sheep is similar to BSE-L with regards to incubation period, attack rate, and vacuolation profile. Our findings are in agreement with previous research that found phenotypic similarities between
<figure><img src="image_36.png" /></figure>

However, the normal function(s) of PrPC<sup>C</sup> are not well understood. We have used our prp1; prp2 compound homozygous knockout mutant zebrafish to identify potentially conserved, cross-species functions of PrPC<sup>C</sup> using RNA-sequencing analysis in addition to exploring the increasing evidence for PrPC<sup>C</sup> involvement in cell adhesion.

**Methods:** Three-day post fertilization wild-type and prp1<sup>ua5003/ua5003</sup>; prp2<sup>ua5001/ua5001</sup> zebrafish larvae underwent PE100-125 HiDeq2500 RNA-sequencing. Three biological replicates each consisting of 50 pooled larvae were used for each genotype. Analysis of sequencing results was processed using Geneious R9/10 and R packages cufflinks and cummeRbund. Alterations in transcript abundance were confirmed using RT-qPCR for selected genes of interest. Further cell adhesion studies were carried out through *in situ* hybridization analysis and immunohistochemistry.

**Results:** RNA-sequencing analysis of mutant fish show large changes in transcript abundance (log<sub>2</sub> fold change of 0.5 or greater) of 1,249 genes compared to wild-type fish. Of interest are genes involved in cell adhesion, including *st8sia2*, *ncam1a* and members of the protocadherin family, which are thought to be involved during early development and continued maintenance of the central nervous system (CNS). Through search and comparison with published transcriptomes from embryonic PrPC<sup>C</sup> knockout mice, we have identified many common affected biological processes in our mutant zebrafish including apoptosis, proteolysis, oxidative stress and cell proliferation, and adhesion. Further analysis on cell adhesion through RT-qPCR of *ncam1a* and *st8sia2* shows a significant reduction in transcript abundance between wild-type and mutant fish, with *in situ* hybridization of *ncam1a* confirming a decreased distribution of mRNA across the CNS.

**Conclusions:** Our transcriptomic sequencing analysis supports a role for PrPC<sup>C</sup> during early development and sustainment of the CNS, seen through significant changes in transcript abundance in members of the protocadherin family and other genes responsible for cell adhesion and proliferation. We propose cell adhesion is an evolutionary conserved function of PrPC<sup>C</sup> which will be of important consideration regarding how PrPC<sup>C</sup> function may be affected during disease.

**KEYWORDS** Cellular prion protein; cell adhesion; RNA-sequencing; zebrafish

49. The agent of transmissible mink encephalopathy
passaged in sheep is similar to BSE-L

E. D. Cassmann<sup>a,b</sup>, S. J. Moore<sup>a,b</sup>, R. D. Kokemuller<sup>a</sup>, A. Balkema-Buschmann<sup>c</sup>, M. H. Groschup<sup>c</sup> and J. J. Greenlee<sup>a</sup>

<sup>a</sup>Virus and Prion Research Unit, National Animal Disease Center, ARS, United States Department of Agriculture, Ames, IA, USA (EDC, SJM, RDK, JJG); <sup>b</sup>Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the U.S. Department of Energy (DOE) and the U.S. Department of Agriculture (USDA). ORISE is managed by ORAU under DOE contract number DE-SC0014664. (EDC, SJM), Department of Veterinary Pathology, Iowa State University, Ames, IA, USA (JDS); <sup>c</sup>Institute of Novel and Emerging Infectious Diseases, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Greifswald - Isle of Riems, Germany (ABB, MHG)

CONTACT E. D. Cassmann eric.cassmann@usda.gov

ABSTRACT

**Introduction:** Transmissible mink encephalopathy (TME) is a fatal neurologic prion disease of farmed mink. Epidemiologic and experimental evidence following a Wisconsin outbreak in 1985 has linked TME to low-type bovine spongiform encephalopathy (BSE-L). Evidence suggests that farmed mink were likely exposed through feeding of BSE-L infected downer cattle. The interspecies transmission of TME to cattle has been documented. Recently, we demonstrated the susceptibility of sheep to cattle passaged TME by intracranial inoculation. The aim of the present study was to compare ovine passaged cattle TME to other prion diseases of food-producing animals. Using a bovine transgenic mouse model, we compared the disease phenotype of sheep TME to BSE-C and BSE-L.

**Materials and Methods:** Separate inoculants of sheep passaged TME were derived from animals with the VRQ/VRQ (VV<sub>136</sub>) and ARQ/VRQ (AV<sub>136</sub>) prion protein genotype. Transgenic bovinized mice (TgBovXV) were intracranially inoculated with 20 µl of 1% w/v brain homogenate. The disease phenotypes were characterized by comparing the attack rates, incubation periods, and vacuolation profiles in TgBovXV mice.

**Results:** The attack rate for BSE-C (13/13), BSE-L (18/18), and TME<sub>VV</sub> (21/21) was 100%; whereas, the TME<sub>AV</sub> group (15/19) had an incomplete attack rate. The average incubation periods were 299, 280, 310, and 541 days, respectively. The vacuolation profiles of BSE-L and TME<sub>VV</sub> were most similar with mild differences observed in the thalamus and medulla. Vacuolation profiles from the BSE-C and TME<sub>AV</sub> experimental groups were different than TME<sub>VV</sub> and BSE-L.

**Conclusion:** Overall the phenotype of disease in TME inoculated transgenic mice was dependent on the sheep donor genotype (VV vs AV). The results of the present study indicate that TME isolated from VRQ/VRQ sheep is similar to BSE-L with regards to incubation period, attack rate, and vacuolation profile. Our findings are in agreement with previous research that found phenotypic similarities between
<figure><img src="image_33.png" /></figure>

BSE-L and cattle passaged TME in an ovine trans-
genic rodent model. In this study, the similarities
between ovine TME and BSE-L are maintained after
multiple interspecies passages.

**50. Cellular Prion protein on human leucocytes is associated with iron metabolism**

Ewald Lindner<sup>a</sup>, Nora Woltsche<sup>a</sup>, Domagoj Ivastinovic<sup>a</sup>, Verena Zrim<sup>b</sup>, Rita Moos<sup>c</sup>, Andreas Wedrich<sup>a</sup> and Adriano Aguzzi<sup>c</sup>

<sup>a</sup>Department of Ophthalmology, Medical University Graz; <sup>b</sup>Core Facility Imaging, Center for Medical Research Graz; <sup>c</sup>Institute of Neuropathology, University Hospital Zurich

CONTACT Ewald Lindner Ewald.lindner@medunigraz.at

ABSTRACT

**Introduction:** The function of the prion protein
remains elusive. Several findings indicate a role in
iron metabolism. In this study, we wanted to investigate
an association between indicators of iron storage and
the expression of prion protein on human leucocytes in
glaucoma patients.

**Materials and Methods:** Patients were recruited at the Department of Ophthalmology at the Medical University Graz. Twenty patients with glaucoma were included. The expression of prion protein on CD3+ CD4+, CD3+ CD8+ and CD14+ leukocytes was investigated by FACS with POM1 antibody.

**Results:** The expression of Prion Protein correlated significantly with soluble transferrin receptor, haemo-globin, and serum iron (Pearson *r* > 0.6; *p*-value < 0.01). No association was found between expression of prion protein and glaucoma progression.

**Conclusions:** We found a strong correlation between iron metabolism and the expression of prion protein on human leucocytes. This presents add to our understanding of the function and regulation of the cellular prion protein.

**KEYWORDS:** Prion protein; human leucocytes; soluble transferrin receptor; haemoglobin

**51. Cryptic prion strains/variants appear when anti-prion systems are disabled**

Reed B. Wickner, Moonil Son, Evgeny Bezsonov,
Songsong Wu, Morgan Dewilde and Herman K. Edskes

Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA

CONTACT Reed B. Wickner wickner@helix.nih.gov

ABSTRACT

The yeast prions [PSI+] and [URE3] are in-register
parallel amyloids of Sup35p and Ure2p, respectively.
Ssb1,2p are ribosome-associated Hsp70s that ~10-fold
repress [PSI+] generation. Upf1,2,3p are factors
essential for mRNA nonsense-mediated decay that,
at normal levels, cure >90% of [PSI+] variants arising
in any *upf* mutant. The formation of the Upf1,2,3-
Sup35 complex is critical for the curing process. The
*upf* mutants show 10–15 fold elevation in frequency
of spontaneous or induced [PSI+] generation. By
controlling cellular levels of 5-pyrophospho-inositol
pentaphosphate, Siw14p prevents the propagation of
about half of the [PSI+] variants arising in its
absence. Normal levels of the disaggregating chaper-
one Hsp104 cures about half of the [PSI+] variants
that arise in an *hsp104*<sup>T160M</sup> mutant defective in the
Hsp104 overproduction curing activity. Normal levels
of Bs2p and Cur1p are paralogs that cure ~90% of
[URE3] prion strains/variants arising in a *btn2Δ
cur1Δ* strain, preferentially those with low propagon
numbers. Bs2p (but not Cur1p) cures by collecting
Ure2p amyloid filaments at a single site in the cell
co-inciding with Bs2p itself. Cell division results in
frequent curing of one of the daughter cells.

The common thread of most of these systems is that
prion strains that would not survive in a wild type
strain are detected in mutants defective in one or
more of these anti-prion systems. We infer that the
normal cell experiences a large load of nascent prions,
most of which are quickly eliminated by one or more of
these systems. The mechanistic details, insofar as we
know them, argue that each of these systems are dis-
tinct. The excess prion strains/variants detected in cells
defective in one anti-prion system are evidently
immune to curing by the other anti-prion systems,
indicating that each anti-prion system defines a new
class of prion strains/variants. It is likely that some
prion/prion-like diseases are a consequence of break-
down of homologous/analogous human anti-prion
systems.

Future work will address the following questions:

* What other components are involved in each of these processes? Is there any overlap?

* Do cells defective in multiple anti-prion systems show even higher prion generation frequencies?

* What other anti-prion systems are there?
<figure><img src="image_37.png" /></figure>

BSE-L and cattle passaged TME in an ovine trans-
genic rodent model. In this study, the similarities
between ovine TME and BSE-L are maintained after
multiple interspecies passages.

50. Cellular Prion protein on human leucocytes is associated with iron metabolism

Ewald Lindner<sup>a</sup>, Nora Woltsche<sup>a</sup>, Domagoj Ivastinovic<sup>a</sup>, Verena Zrim<sup>b</sup>, Rita Moos<sup>c</sup>, Andreas Wedrich<sup>a</sup> and Adriano Aguzzi<sup>c</sup>

<sup>a</sup>Department of Ophthalmology, Medical University Graz; <sup>b</sup>Core Facility Imaging, Center for Medical Research Graz; <sup>c</sup>Institute of Neuropathology, University Hospital Zurich

CONTACT Ewald Lindner Ewald.lindner@medunigraz.at

ABSTRACT

**Introduction:** The function of the prion protein
remains elusive. Several findings indicate a role in
iron metabolism. In this study, we wanted to investigate
an association between indicators of iron storage and
the expression of prion protein on human leucocytes in
glaucoma patients.

**Materials and Methods:** Patients were recruited at the Department of Ophthalmology at the Medical University Graz. Twenty patients with glaucoma were included. The expression of prion protein on CD3+ CD4+, CD3+ CD8+ and CD14+ leukocytes was investigated by FACS with POM1 antibody.

**Results:** The expression of Prion Protein correlated significantly with soluble transferrin receptor, haemo-globin, and serum iron (Pearson *r* > 0.6; *p*-value < 0.01). No association was found between expression of prion protein and glaucoma progression.

**Conclusions:** We found a strong correlation between iron metabolism and the expression of prion protein on human leucocytes. This presents add to our understanding of the function and regulation of the cellular prion protein.

**KEYWORDS:** Prion protein; human leucocytes; soluble transferrin receptor; haemoglobin

**51. Cryptic prion strains/variants appear when anti-prion systems are disabled**

Reed B. Wickner, Moonil Son, Evgeny Bezsonov,
Songsong Wu, Morgan Dewilde and Herman K. Edskes

Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA

CONTACT Reed B. Wickner wickner@helix.nih.gov

ABSTRACT

The yeast prions [PSI+] and [URE3] are in-register
parallel amyloids of Sup35p and Ure2p, respectively.
Ssb1,2p are ribosome-associated Hsp70s that ~10-fold
repress [PSI+] generation. Upf1,2,3p are factors
essential for mRNA nonsense-mediated decay that,
at normal levels, cure >90% of [PSI+] variants arising
in any *upf* mutant. The formation of the Upf1,2,3-
Sup35 complex is critical for the curing process. The
*upf* mutants show 10–15 fold elevation in frequency
of spontaneous or induced [PSI+] generation. By
controlling cellular levels of 5-pyrophospho-inositol
pentaphosphate, Siw14p prevents the propagation of
about half of the [PSI+] variants arising in its
absence. Normal levels of the disaggregating chaper-
one Hsp104 cures about half of the [PSI+] variants
that arise in an *hsp104*<sup>T160M</sup> mutant defective in the
Hsp104 overproduction curing activity. Normal levels
of Bs2p and Cur1p are paralogs that cure ~90% of
[URE3] prion strains/variants arising in a *btn2Δ
cur1Δ* strain, preferentially those with low propagon
numbers. Bs2p (but not Cur1p) cures by collecting
Ure2p amyloid filaments at a single site in the cell
co-inciding with Bs2p itself. Cell division results in
frequent curing of one of the daughter cells.

The common thread of most of these systems is that
prion strains that would not survive in a wild type
strain are detected in mutants defective in one or
more of these anti-prion systems. We infer that the
normal cell experiences a large load of nascent prions,
most of which are quickly eliminated by one or more of
these systems. The mechanistic details, insofar as we
know them, argue that each of these systems are dis-
tinct. The excess prion strains/variants detected in cells
defective in one anti-prion system are evidently
immune to curing by the other anti-prion systems,
indicating that each anti-prion system defines a new
class of prion strains/variants. It is likely that some
prion/prion-like diseases are a consequence of break-
down of homologous/analogous human anti-prion
systems.

Future work will address the following questions:

* What other components are involved in each of these processes? Is there any overlap?

* Do cells defective in multiple anti-prion systems show even higher prion generation frequencies?

* What other anti-prion systems are there?
<figure><img src="image_34.png" /></figure>

**52. Microglial-specific exosome isolation from prion-infected fluids**

Stephanie Booth, Lise Lamoureux and Sarah Medina

Public Health Agency of Canada, NML

**ABSTRACT**

Prion disease is caused by the conversion of $PrP^c$ (cel-
lular prion protein) to $PrP^{sc}$ (abnormal prion protein)
which aggregates in the brain causing spongiform neu-
rodegeneration. In response, astrocytes and microglia
are activated. Both $PrP^c$ and $PrP^{sc}$ have been shown to
be associated with exosomes, which are thought to be
involved in prion pathogenesis. Exosomes are small 30–
150nm vesicles that are released from cells via exocy-
tosis into biological fluids. They are involved in cell to
cell communication as they can cross biological barriers
easily. Exosomes contain protein, lipids, and nucleic
acids that are representative of the cell types from
which they originate. Therefore, they could be specifi-
cally isolated and used as the basis for cell-type specific
biomarker discovery. Few studies have looked at prion-
specific changes in the expression of biomolecules in
exosomes from cells that are involved in the disease
process. Exosomes originating from the brain (specifi-
cally microglia) could be an ideal source for disease-
specific molecular signatures from early stages of dis-
ease. We hypothesized that TMEM119, a transmem-
brane protein that is expressed highly specific on
microglial cells, could be used as a marker to specifi-
cally select microglial exosomes. The goal of this pre-
liminary study was to develop methods to isolate
microglial-specific exosomes from cells infected with
prions. We used western blots and nanoparticle track-
ing analysis (NTA) to look at the composition of
microglial-specific exosomes. Exosomes were isolated
in media from BV2 cells (a microglial cell line). A
TMEM119 antibody was labelled with a fluorophore
and binding to exosomes was visualized via NTA.
Exosomes were also isolated from prion-infected and
uninfected primary cerebellum slice cultures.
TMEM119 antibody was bound to beads which could
then successfully ‘pull-out’ microglial specific exo-
somes. This methodology will be tested for its utility
in prion disease-specific biomarker discovery from
microglial exosomes isolated from biofluids from
infected animals.

**53. Evaluation of the inter-species transmission potential of different CWD isolates**

Rodrigo Morales<sup>a</sup>, Carlos Kramm<sup>a,b</sup>, Paulina Soto<sup>a</sup>,
Adam Lyon<sup>a</sup>, Sandra Pritzkow<sup>a</sup>, Claudio Soto<sup>a</sup>

<sup>a</sup>Mitchell Center for Alzheimer's disease and Related Brain Disorders, Dept. of Neurology, McGovern School of Medicine University of Texas Health Science Center at Houston, TX, USA; <sup>b</sup>Facultad de Medicina, Universidad de los Andes, Santiago, Chile

**ABSTRACT**

Chronic Wasting Disease (CWD) has reached epidemic proportions in North America and has been identified in South Korea and Northern Europe. CWD-susceptible cervid species are known to share habitats with humans and other animals entering the human food chain. At present, the potential of CWD to infect humans and other animal species is not completely clear. The exploration of this issue acquires further complexity considering the differences in the prion protein sequence due to species-specific variations and polymorphic changes within species. While several species of cervids are naturally affected by CWD, white-tailed deer (WTD) is perhaps the most relevant due to its extensive use in hunting and as a source of food. Evaluation of inter-species prion infections using animals or mouse models is costly and time consuming. We and others have shown that the Protein Misfolding Cyclic Amplification (PMCA) technology reproduces, in an accelerated and inexpensive manner, the inter-species transmission of prions while preserving the strain features of the input $PrP^{Sc}$. In this work, we tested the potential of different WTD-derived CWD isolates to transmit to humans and other animal species relevant for human consumption using PMCA. For these experiments, CWD isolates homozygous for the most common WTD-PrP polymorphic changes (G96S) were used (96SS variant obtained from a pre-symptomatic prion infected WTD). Briefly, 96GG and 96SS CWD prions were adapted in homologous or heterologous substrate by PMCA through several (15) rounds. End products, as well as intermediates across the process, were tested for their inter-species transmission potentials. A similar process was followed to assess seed-templated misfolding of ovine, porcine, and bovine $PrP^C$. Our results show differences on the inter-species transmission potentials of the four adapted materials generated ($PrP^C/PrP^{Sc}$ polymorphic combinations), being the homologous combinations of seed/substrate the ones with the greater apparent zoonotic potential. Surprisingly, 96SS prions adapted in homologous substrate were the ones showing the easiest potential to template $PrP^C$ misfolding from other animal species. In summary, our results show that a plethora of different CWD isolates, each comprising different potentials for inter-species transmission, may exist in the environment. These experiments may help to clarify an uncertain and potentially worrisome public health issue. Additional research in this area may be
<figure><img src="image_38.png" /></figure>

**52. Microglial-specific exosome isolation from prion-infected fluids**

Stephanie Booth, Lise Lamoureux and Sarah Medina

Public Health Agency of Canada, NML

**ABSTRACT**

Prion disease is caused by the conversion of $PrP^c$ (cel-
lular prion protein) to $PrP^{sc}$ (abnormal prion protein)
which aggregates in the brain causing spongiform neu-
rodegeneration. In response, astrocytes and microglia
are activated. Both $PrP^c$ and $PrP^{sc}$ have been shown to
be associated with exosomes, which are thought to be
involved in prion pathogenesis. Exosomes are small 30–
150nm vesicles that are released from cells via exocy-
tosis into biological fluids. They are involved in cell to
cell communication as they can cross biological barriers
easily. Exosomes contain protein, lipids, and nucleic
acids that are representative of the cell types from
which they originate. Therefore, they could be specifi-
cally isolated and used as the basis for cell-type specific
biomarker discovery. Few studies have looked at prion-
specific changes in the expression of biomolecules in
exosomes from cells that are involved in the disease
process. Exosomes originating from the brain (specifi-
cally microglia) could be an ideal source for disease-
specific molecular signatures from early stages of dis-
ease. We hypothesized that TMEM119, a transmem-
brane protein that is expressed highly specific on
microglial cells, could be used as a marker to specifi-
cally select microglial exosomes. The goal of this pre-
liminary study was to develop methods to isolate
microglial-specific exosomes from cells infected with
prions. We used western blots and nanoparticle track-
ing analysis (NTA) to look at the composition of
microglial-specific exosomes. Exosomes were isolated
in media from BV2 cells (a microglial cell line). A
TMEM119 antibody was labelled with a fluorophore
and binding to exosomes was visualized via NTA.
Exosomes were also isolated from prion-infected and
uninfected primary cerebellum slice cultures.
TMEM119 antibody was bound to beads which could
then successfully ‘pull-out’ microglial specific exo-
somes. This methodology will be tested for its utility
in prion disease-specific biomarker discovery from
microglial exosomes isolated from biofluids from
infected animals.

**53. Evaluation of the inter-species transmission potential of different CWD isolates**

Rodrigo Morales<sup>a</sup>, Carlos Kramm<sup>a,b</sup>, Paulina Soto<sup>a</sup>,
Adam Lyon<sup>a</sup>, Sandra Pritzkow<sup>a</sup>, Claudio Soto<sup>a</sup>

<sup>a</sup>Mitchell Center for Alzheimer's disease and Related Brain Disorders, Dept. of Neurology, McGovern School of Medicine University of Texas Health Science Center at Houston, TX, USA; <sup>b</sup>Facultad de Medicina, Universidad de los Andes, Santiago, Chile

**ABSTRACT**

Chronic Wasting Disease (CWD) has reached epidemic proportions in North America and has been identified in South Korea and Northern Europe. CWD-susceptible cervid species are known to share habitats with humans and other animals entering the human food chain. At present, the potential of CWD to infect humans and other animal species is not completely clear. The exploration of this issue acquires further complexity considering the differences in the prion protein sequence due to species-specific variations and polymorphic changes within species. While several species of cervids are naturally affected by CWD, white-tailed deer (WTD) is perhaps the most relevant due to its extensive use in hunting and as a source of food. Evaluation of inter-species prion infections using animals or mouse models is costly and time consuming. We and others have shown that the Protein Misfolding Cyclic Amplification (PMCA) technology reproduces, in an accelerated and inexpensive manner, the inter-species transmission of prions while preserving the strain features of the input $PrP^{Sc}$. In this work, we tested the potential of different WTD-derived CWD isolates to transmit to humans and other animal species relevant for human consumption using PMCA. For these experiments, CWD isolates homozygous for the most common WTD-PrP polymorphic changes (G96S) were used (96SS variant obtained from a pre-symptomatic prion infected WTD). Briefly, 96GG and 96SS CWD prions were adapted in homologous or heterologous substrate by PMCA through several (15) rounds. End products, as well as intermediates across the process, were tested for their inter-species transmission potentials. A similar process was followed to assess seed-templated misfolding of ovine, porcine, and bovine $PrP^C$. Our results show differences on the inter-species transmission potentials of the four adapted materials generated ($PrP^C/PrP^{Sc}$ polymorphic combinations), being the homologous combinations of seed/substrate the ones with the greater apparent zoonotic potential. Surprisingly, 96SS prions adapted in homologous substrate were the ones showing the easiest potential to template $PrP^C$ misfolding from other animal species. In summary, our results show that a plethora of different CWD isolates, each comprising different potentials for inter-species transmission, may exist in the environment. These experiments may help to clarify an uncertain and potentially worrisome public health issue. Additional research in this area may be
<figure><img src="image_35.png" /></figure>

useful to advise on the design of regulations intended to stop the spread of CWD and predict unwanted zoonotic events.

## 54. Analysis of amyloid properties of MBP protein in the brain of rat R. norvegicus

A. A. Shenfeld<sup>a,b*</sup>, M. J. Velizhanina<sup>a</sup>, J. V. Sopova<sup>a,b</sup>, T. A. Belashova<sup>b</sup> and A. P. Galkin<sup>a,b</sup>

<sup>a</sup>St-Petersburg State University, dept of Genetics and Biotechnology, St-Petersburg, Russia; <sup>b</sup>St-Petersburg branch of Vavilov Institute of General Genetics, Lab. of Genetic Modeling of Human Diseases, St-Petersburg, Russia

CONTACT A. A. Shenfeld shenaleksandr@mail.ru

## 55. PAD-beads enhances detection of $PrP^{Sc}$ in scrapie infected sheep brain samples using real-time quaking-induced conversion

Soyoun Hwang, Rohana P. Dassanayake and Eric M. Nicholson

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

CONTACT Soyoun Hwang Soyoun.Hwang@ars.usda.gov

### ABSTRACT

Scrapie, a transmissible spongiform encephalopathy (TSE), naturally occurs in sheep and goats. This fatal neurodegenerative disease results from misfolding of the cellular prion protein ($PrP^C$) to a pathogenic prion protein form ($PrP^{Sc}$). This pathogenic form accumulates in the brain and other tissues. The presence of $PrP^{Sc}$ can be detected by an *in vitro* conversion assay known as real-time quaking induced conversion (RT-QuIC). RT-QuIC has been used to detect $PrP^{Sc}$ in a variety of biological tissues from brains to fluids. While this technique is both rapid and sensitive enhancing the detection of prions would be valuable in the diagnostic laboratories. In this study, we evaluated if RT-QuIC can be coupled to $PrP^{Sc}$ binding to a $PrP^{Sc}$ specific ligand found on commercially available PAD-Beads for clean up of $PrP^{Sc}$ containing samples enhanced prion detection. Coupling the RT-QuIC amplification to PAD-Bead based cleanup allowed detection of scrapie prion rapidly and without dilutions sheep brain homogenates prior to RT-QuIC. The PAD-Bead sample pretreatment step prior to RT-QuIC is a useful enhancement in the diagnosis of TSEs.

**KEYWORDS:** Scrapie; prion diseases; RT-QuIC; transmissible spongiform encephalopathy; PAD-Beads; magnetic particle extraction

## 56. Understanding chronic wasting disease spread potential for at-risk species

Catherine I. Cullingham, Anh Dao, Debbie McKenzie and David W. Coltman

Department of Biological Sciences, University of Alberta, Edmonton AB, Canada

CONTACT Catherine I. Cullingham cathy.cullingham@ualberta.ca

### ABSTRACT

Genetic variation can be linked to susceptibility or resistance to a disease, and this information can help to better understand spread-risk in a population. Wildlife disease incidence is increasing, and this is resulting in negative impacts on the economy, biodiversity, and in some instances, human health. If we can find genetic variation that helps to inform which individuals are susceptible, then we can use this information on at-risk populations to better manage negative consequences. Chronic wasting disease, a fatal, transmissible spongiform encephalopathy of cervids (both wild and captive), continues to spread geographically, which has resulted in an increasing host-range. The disease agent ($PrP^{CWD}$) is a misfolded conformer of native cellular protein ($PrP^C$). In Canada, the disease is endemic in Alberta and Saskatchewan, infecting primarily mule deer and white-tail deer, with a smaller impact on elk and moose populations. As the extent of the endemic area continues to expand, additional species will be exposed to this disease, including bison, bighorn sheep, mountain goat, and pronghorn antelope. To better understand the potential spread-risk among these species, we reviewed the current literature on species that have been orally exposed to CWD to identify susceptible and resistant species. We then compared the amino acid polymorphisms of $PrP^C$ among these species to determine whether any sites were linked to susceptibility or resistance to CWD infection. We sequenced the entire $PrP$ coding region in 578 individuals across at-risk populations to evaluate their potential susceptibility. Three amino acid sites (97, 170, and 174; human numbering) were significantly associated with susceptibility, but these were not fully discriminating. All but one species among the resistant group shared the same haplotype, and the same for the susceptible species. For the at-risk species, bison had the resistant haplotype, while bighorn sheep and mountain goats were closely associated with the resistant type. Pronghorn antelope and a newly identified haplotype in moose differed from the susceptible haplotype, but were still closely associated with it. These data suggest pronghorn antelope will be susceptible to CWD while bison are likely to be resistant. Based on this data, recommendations can be made regarding species to be monitored for possible CWD infection.
<figure><img src="image_39.png" /></figure>

useful to advise on the design of regulations intended to stop the spread of CWD and predict unwanted zoonotic events.

## 54. Analysis of amyloid properties of MBP protein in the brain of rat R. norvegicus

A. A. Shenfeld<sup>a,b*</sup>, M. J. Velizhanina<sup>a</sup>, J. V. Sopova<sup>a,b</sup>, T. A. Belashova<sup>b</sup> and A. P. Galkin<sup>a,b</sup>

<sup>a</sup>St-Petersburg State University, dept of Genetics and Biotechnology, St-Petersburg, Russia; <sup>b</sup>St-Petersburg branch of Vavilov Institute of General Genetics, Lab. of Genetic Modeling of Human Diseases, St-Petersburg, Russia

CONTACT A. A. Shenfeld shenaleksandr@mail.ru

## 55. PAD-beads enhances detection of PrP<sup>Sc</sup> in scrapie infected sheep brain samples using real-time quaking-induced conversion

Soyoun Hwang, Rohana P. Dassanayake and Eric M. Nicholson

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

CONTACT Soyoun Hwang Soyoun.Hwang@ars.usda.gov

### ABSTRACT

Scrapie, a transmissible spongiform encephalopathy (TSE), naturally occurs in sheep and goats. This fatal neurodegenerative disease results from misfolding of the cellular prion protein (PrP<sup>C</sup>) to a pathogenic prion protein form (PrP<sup>Sc</sup>). This pathogenic form accumulates in the brain and other tissues. The presence of PrP<sup>Sc</sup> can be detected by an *in vitro* conversion assay known as real-time quaking induced conversion (RT-QuIC). RT-QuIC has been used to detect PrP<sup>Sc</sup> in a variety of biological tissues from brains to fluids. While this technique is both rapid and sensitive enhancing the detection of prions would be valuable in the diagnostic laboratories. In this study, we evaluated if RT-QuIC can be coupled to PrP<sup>Sc</sup> binding to a PrP<sup>Sc</sup> specific ligand found on commercially available PAD-Beads for clean up of PrP<sup>Sc</sup> containing samples enhanced prion detection. Coupling the RT-QuIC amplification to PAD-Bead based cleanup allowed detection of scrapie prion rapidly and without dilutions sheep brain homogenates prior to RT-QuIC. The PAD-Bead sample pretreatment step prior to RT-QuIC is a useful enhancement in the diagnosis of TSEs.

**KEYWORDS:** Scrapie; prion diseases; RT-QuIC; transmissible spongiform encephalopathy; PAD-Beads; magnetic particle extraction

## 56. Understanding chronic wasting disease spread potential for at-risk species

Catherine I. Cullingham, Anh Dao, Debbie McKenzie and David W. Coltman

Department of Biological Sciences, University of Alberta, Edmonton AB, Canada

CONTACT Catherine I. Cullingham cathy.cullingham@ualberta.ca

### ABSTRACT

Genetic variation can be linked to susceptibility or resistance to a disease, and this information can help to better understand spread-risk in a population. Wildlife disease incidence is increasing, and this is resulting in negative impacts on the economy, biodiversity, and in some instances, human health. If we can find genetic variation that helps to inform which individuals are susceptible, then we can use this information on at-risk populations to better manage negative consequences. Chronic wasting disease, a fatal, transmissible spongiform encephalopathy of cervids (both wild and captive), continues to spread geographically, which has resulted in an increasing host-range. The disease agent (PrP<sup>CWD</sup>) is a misfolded conformer of native cellular protein (PrP<sup>C</sup>). In Canada, the disease is endemic in Alberta and Saskatchewan, infecting primarily mule deer and white-tail deer, with a smaller impact on elk and moose populations. As the extent of the endemic area continues to expand, additional species will be exposed to this disease, including bison, bighorn sheep, mountain goat, and pronghorn antelope. To better understand the potential spread-risk among these species, we reviewed the current literature on species that have been orally exposed to CWD to identify susceptible and resistant species. We then compared the amino acid polymorphisms of PrP<sup>C</sup> among these species to determine whether any sites were linked to susceptibility or resistance to CWD infection. We sequenced the entire PrP coding region in 578 individuals across at-risk populations to evaluate their potential susceptibility. Three amino acid sites (97, 170, and 174; human numbering) were significantly associated with susceptibility, but these were not fully discriminating. All but one species among the resistant group shared the same haplotype, and the same for the susceptible species. For the at-risk species, bison had the resistant haplotype, while bighorn sheep and mountain goats were closely associated with the resistant type. Pronghorn antelope and a newly identified haplotype in moose differed from the susceptible haplotype, but were still closely associated with it. These data suggest pronghorn antelope will be susceptible to CWD while bison are likely to be resistant. Based on this data, recommendations can be made regarding species to be monitored for possible CWD infection.
<figure><img src="image_36.png" /></figure>

**KEYWORDS:** Chronic wasting disease; Prnp; wildlife disease; population genetics; ungulates

**57. Oligomerization of Aβ(1–40/42) in the presence of somatostatin-14 investigated in silico**

Min Wu<sup>a</sup>, Lyudmyla Dorosh<sup>a,b</sup>, Gerold Schmitt-Ulms<sup>c</sup>, Holger Wille<sup>d,e</sup>, Maria Stepanova<sup>a,b</sup>

<sup>a</sup>Department of Electrical and Computer Engineering, University of Alberta, Edmonton, Canada; <sup>b</sup>National Research Council of Canada, Edmonton, Alberta, Canada; <sup>c</sup>Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada; <sup>d</sup>Department of Biochemistry, University of Alberta, Edmonton, Canada; <sup>d</sup>Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Canada

CONTACT Min Wu wu2@ualberta.ca; Lyudmyla Dorosh dorosh@ualberta.ca

**ABSTRACT**

Alzheimer's disease (AD) is associated with pathological oligomeric aggregates and fibrils formed by amyloid beta (Aβ) peptides. Experiments indicated that somatostatin-14 (SST₁₄), a small cyclic peptide found in the brain, may influence the formation of toxic oligomers of Aβ₁⁻₄₂, but not Aβ₁⁻₄₀ [1]. However, the specific role of SST₁₄ and the reason for the different responses of Aβ₄₂ and Aβ₄₀ remain unclear. We have investigated the effects of SST₁₄ on aggregation of Aβ₁⁻₄₀ and Aβ₁⁻₄₂ peptides using 100 ns long all-atom molecular dynamics simulations. Starting from randomly positioned Aβ₄₀/₄₂ and SST₁₄ chains in water, we observed the formation of oligomers, and investigated their structure and dynamics. For comparison, we have investigated a similar system containing Aβ₁⁻₄₂ and the arginine vasopressin (AVP) peptide as a negative control [1]. We have employed well-established structural biology tools, as well as a novel essential collective dynamics (ECD) method [2], which allows analysing pair correlations, main-chain flexibilities, and domains of correlated motion within the same framework. We observed formation of stable co-oligomers containing Aβ chains and SST₁₄ or AVP molecules in all the three systems, Aβ₄₂-SST₁₄, Aβ₄₀-SST₁₄, and Aβ₄₂-AVP. During the simulations, the number of hydrogen bonds in the systems gradually increased. This was accompanied by a buildup of salt bridges, hydrophobic contacts, and other interactions responsible for the oligomerization process. Interestingly, C-terminal residues of both Aβ₄₂ and Aβ₄₀ are often located at the surface of the oligomers, whereas their central parts stay preferentially in the interior regions. The ECD analysis indicated that in most Aβ oligomers, C-termini exhibited greater main-chain flexibility in comparison to central regions, also indicating a stronger solvent

exposure of the C-termini. The solvent accessible surface area (SASA) of hydrophobic side groups is the greatest in Aβ₄₂-SST₁₄ oligomers, in part due to the larger hydrophobic SASA of SST₁₄ in comparison to the AVP control. Aβ₄₂-AVP oligomers are relatively small in size, and their hydrophobic SASA is less than in the other systems. We conclude that differences in aggregation of Aβ₄₂-SST₁₄ and the other two systems may result from a greater hydrophobic SASA of both Aβ₄₂ and SST₁₄, producing a stronger ‘sticky surface’ effect. In Aβ₄₂-AVP the effect appears lessened by a lower hydrophobicity of AVP, whereas in Aβ₄₀-SST₁₄, the reduced number of solvent-exposed hydrophobic C-terminal residues of Aβ may play a role.

**References**

[1] Wang H, Muiznieks LD, Ghosh P, et al. Somatostatin binds to the human amyloid β peptide and favors the formation of distinct oligomers. *eLife*. 2017;6:e28401.

[2] Dorosh L, Stepanova M. Probing oligomerization of amyloid beta peptide in silico. *Mol.BioSyst.* 2017;13:165–182.

**58. Understanding the mechanisms and effects of impaired Rab7 function in prion infection**

Pearl Cherry, Su Yeon Shim and Sabine Gilch

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

CONTACT Pearl Cherry pearl.cherry@ucalgary.ca

**ABSTRACT**

**Background:** In persistently prion-infected cell cultures, reduced membrane association of Rab7 and, as a consequence, reduced lysosomal maturation and impaired lysosomal degradation is observed. Other hallmarks of prion infections are increased cholesterol levels and impaired axonal retrograde trafficking. Targeted cargo trafficking is regulated mainly by rab proteins and requires them to shuttle between an active GTP and membrane bound and an inactive cytosolic state. Rab7 is a protein essential for intracellular transport of cargos like low density lipoprotein (LDL) and neurotrophins. The aim of this study is to elucidate the causes and effects of reduced membrane bound Rab7, specifically on how it influences PrPSc propagation, trafficking of low density lipoprotein (LDL) and neurotrophins and hence its effect on cholesterol metabolism and neurodegeneration.

**Methods:** We used prion infected (strain 22L) N2a cells for transient transfection with different functional mutants of Rab7 including the wild type (WT), the constitutively active
<figure><img src="image_40.png" /></figure>

**KEYWORDS:** Chronic wasting disease; Prnp; wildlife disease; population genetics; ungulates

**57. Oligomerization of Aβ(1-40/42) in the presence of somatostatin-14 investigated in silico**

Min Wuª, Lyudmyla Doroshª,ᵇ, Gerold Schmitt-Ulmsᶜ, Holger Willed,ᵉ, Maria Stepanovaª,ᵇ

ªDepartment of Electrical and Computer Engineering, University of Alberta, Edmonton, Canada; ᵇNational Research Council of Canada, Edmonton, Alberta, Canada; ᶜLaboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada; ᵈDepartment of Biochemistry, University of Alberta, Edmonton, Canada; ᵉCentre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Canada

**CONTACT** Min Wu wu2@ualberta.ca; Lyudmyla Dorosh dorosh@ualberta.ca

**ABSTRACT**

Alzheimer's disease (AD) is associated with pathological oligomeric aggregates and fibrils formed by amyloid beta (Aβ) peptides. Experiments indicated that somatostatin-14 (SST₁₄), a small cyclic peptide found in the brain, may influence the formation of toxic oligomers of Aβ₁₋₄₂, but not Aβ₁₋₄₀ [1]. However, the specific role of SST₁₄ and the reason for the different responses of Aβ₄₂ and Aβ₄₀ remain unclear. We have investigated the effects of SST₁₄ on aggregation of Aβ₁₋₄₀ and Aβ₁₋₄₂ peptides using 100 ns long all-atom molecular dynamics simulations. Starting from randomly positioned Aβ₄₀/₄₂ and SST₁₄ chains in water, we observed the formation of oligomers, and investigated their structure and dynamics. For comparison, we have investigated a similar system containing Aβ₁₋₄₂ and the arginine vasopressin (AVP) peptide as a negative control [1]. We have employed well-established structural biology tools, as well as a novel essential collective dynamics (ECD) method [2], which allows analysing pair correlations, main-chain flexibilities, and domains of correlated motion within the same framework. We observed formation of stable co-oligomers containing Aβ chains and SST₁₄ or AVP molecules in all the three systems, Aβ₄₂-SST₁₄, Aβ₄₀-SST₁₄, and Aβ₄₂-AVP. During the simulations, the number of hydrogen bonds in the systems gradually increased. This was accompanied by a buildup of salt bridges, hydrophobic contacts, and other interactions responsible for the oligomerization process. Interestingly, C-terminal residues of both Aβ₄₂ and Aβ₄₀ are often located at the surface of the oligomers, whereas their central parts stay preferentially in the interior regions. The ECD analysis indicated that in most Aβ oligomers, C-termini exhibited greater main-chain flexibility in comparison to central regions, also indicating a stronger solvent

exposure of the C-termini. The solvent accessible surface area (SASA) of hydrophobic side groups is the greatest in Aβ₄₂-SST₁₄ oligomers, in part due to the larger hydrophobic SASA of SST₁₄ in comparison to the AVP control. Aβ₄₂-AVP oligomers are relatively small in size, and their hydrophobic SASA is less than in the other systems. We conclude that differences in aggregation of Aβ₄₂-SST₁₄ and the other two systems may result from a greater hydrophobic SASA of both Aβ₄₂ and SST₁₄, producing a stronger 'sticky surface' effect. In Aβ₄₂-AVP the effect appears lessened by a lower hydrophobicity of AVP, whereas in Aβ₄₀-SST₁₄, the reduced number of solvent-exposed hydrophobic C-terminal residues of Aβ may play a role.

**References**

[1] Wang H, Muiznieks LD, Ghosh P, et al. Somatostatin binds to the human amyloid β peptide and favors the formation of distinct oligomers. eLife. 2017;6:e28401.

[2] Dorosh L, Stepanova M. Probing oligomerization of amyloid beta peptide in silico. Mol.BioSyst. 2017;13:165-182.

**58. Understanding the mechanisms and effects of impaired Rab7 function in prion infection**

Pearl Cherry, Su Yeon Shim and Sabine Gilch

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

**CONTACT** Pearl Cherry pearl.cherry@ucalgary.ca

**ABSTRACT**

**Background:** In persistently prion-infected cell cultures, reduced membrane association of Rab7 and, as a consequence, reduced lysosomal maturation and impaired lysosomal degradation is observed. Other hallmarks of prion infections are increased cholesterol levels and impaired axonal retrograde trafficking. Targeted cargo trafficking is regulated mainly by rab proteins and requires them to shuttle between an active GTP and membrane bound and an inactive cytosolic state. Rab7 is a protein essential for intracellular transport of cargos like low density lipoprotein (LDL) and neurotrophins. The aim of this study is to elucidate the causes and effects of reduced membrane bound Rab7, specifically on how it influences PrPSc propagation, trafficking of low density lipoprotein (LDL) and neurotrophins and hence its effect on cholesterol metabolism and neurodegeneration.

**Methods:** We used prion infected (strain 22L) N2a cells for transient transfection with different functional mutants of Rab7 including the wild type (WT), the constitutively active
<figure><img src="image_37.png" /></figure>

(Q67L) and the transdominant negative (T22N) mutants for 48 h and analysed PrPSc levels. The differential effect of overexpression of these mutants on cholesterol metabolism in 22LN2a cells is analysed through Amplex cholesterol assay. Immunofluorescence experiments are conducted using an antibody against Rab7-GTP in prion infected and non-infected cell lines. Trafficking of LDL is analysed using by pulse-chase experiments with fluorescently labelled LDL and co-localization with endosomal markers.

**Results:** We demonstrate that in the prion infected cell lines, transient over-expression of the constitutively active mutant Rab7 reduces PrPSc propagation. Since prion infected cell cultures exhibit an increase in the cholesterol levels, we analysed how different functional mutants of Rab7 regulate cholesterol levels and observed that over-expression of the constitutively active Q67L mutant reduces the cholesterol level with respect to Rab7-WT mutant in 22LN2a cells. Overexpression of Rab7-WT did neither affect PrPSc levels, nor cholesterol content in those cells. This indicates that activation of Rab7 by GDP/GTP exchange might be impaired in 22LN2a cells. Preliminary results of a comparative analysis of Rab7-GTP in N2a and 22LN2a point at a reduction of Rab7-GTP upon prion infection. Since Rab7 is required for LDL transport, LDL trafficking is analysed to define a link between aberrant vesicle trafficking and enhanced cholesterol synthesis.

**Conclusion:** Through this study, we shed light on the underlying mechanisms and effects of impaired membrane association of Rab7 in prion infection. These effects include deterioration of cellular processes such as vesicle trafficking and cholesterol metabolism, hence favouring PrPSc propagation.

**59. The role of glial cells in BSE pathology: a histomorphological and immunohistochemical investigation**

Christine Fastª, Kristina Santiago-Mateoᵇ, Rakhi Katochᵇ, Martin H. Groschupª, Ute Zieglerª, Stefanie Czubᵇ

ªFriedrich-Loeffler Institut, Isle of Riems, Germany; ᵇCanadian Food Inspection Agency, Lethbridge, Alberta, Canada

CONTACT Christine.Fast@fli.de

ABSTRACT

**Background/Introduction:** Bovine spongiform encephalopathy (BSE) is a fatal neurological disease of cattle associated with the accumulation of an altered form of the cellular prion protein, termed pathological prion protein (PrP<sup>BSE</sup>) The pathomechanisms linking abnormal protein accumulation with the typical severe neurological alterations in BSE are less than clear. Here we investigated the

role of glia cells in the pathology of BSE infected cattle, specifically if and to which degree the histopathological changes are glia mediated and therefore due to the host response and not to the BSE agent itself.

**Material and Methods:** We examined 29 experimentally BSE infected cattle at preclinical (no PrP<sup>BSE</sup> in the brain stem), late preclinical (no clinical signs, first traces of PrP<sup>BSE</sup> in the brain stem), and clinical (clinical signs and distinct accumulation of PrP<sup>BSE</sup> in the brain stem) stages of the disease. The following defined regions of the CNS were examined: Obex, Red Nucleus, Cerebellum, Parietal Cerebrum, Septal Nucleus, and Hippocampus and both histopathological lesions and PrP<sup>BSE</sup> accumulation were determined. Additionally, antibodies Iba-1, CX3CR1, and GFAP were used to visualize and identify glial involvement within the context of the disease. In addition, four unchallenged animals of the same age groups were included as negative controls.

**Results:** PrP<sup>BSE</sup> is found only in neurons and microglia. Different analytic approaches showed that the most consistent changes in glial numbers and phenotypes were detected in the Obex, Red Nucleus, and Hippocampus. However, only in clinical affected cattle, we observed with all antibodies a mild diffuse increase in glial reactivity, indicating a gliosis, as compared to controls, preclinical, and late preclinical animals. Additionally, different glial reaction patterns were almost exclusively detected in clinical animals. This includes a hyperplastic and in parts irregular appearance of the glial network, an accumulation of clumsy cells with shortened processes and rounded cell bodies, a diffusely distributed coarse granular staining and occasionally perineuronal cluster of glial cells.

**Conclusion:** Apart from neurons, we identified microglia as the only other PrP<sup>BSE</sup> accumulating cell population; computerized assessment of IBA1, CX3CR1, and GFAP antibodies are ongoing. The preliminary results presented here indicate that the activation of a glial response is a rather late event, most probably associated with the accumulation of PrP<sup>BSE</sup>.

**60. Caribou Prnp polymorphism distribution in Canada and its impact on CWD pathogenesis**

Maria I. Arifinª, Samia Hannaouiª, Yuan-Hung Huangª, Gordon Mitchellᵇ, Antanas Staskeviciusᵇ, Lech Kaczmarczykᶜ,ᵈ, Walker Jacksonᶜ,ᵈ and Sabine Gilchª

<footnote>ªDepartment of Ecosystem and Public Health, University of Calgary;</footnote>

<footnote>ᵇNational and OIE Reference Laboratory for Scrapie and CWD, Ottawa Laboratory Fallowfield, Canadian Food Inspection Agency;</footnote>

<footnote>ᶜWallenberg Center for Molecular Medicine, Department of Clinical</footnote>
<figure><img src="image_41.png" /></figure>

(Q67L) and the transdominant negative (T22N) mutants for 48 h and analysed PrPSc levels. The differential effect of overexpression of these mutants on cholesterol metabolism in 22LN2a cells is analysed through Amplex cholesterol assay. Immunofluorescence experiments are conducted using an antibody against Rab7-GTP in prion infected and non-infected cell lines. Trafficking of LDL is analysed using by pulse-chase experiments with fluorescently labelled LDL and co-localization with endosomal markers.

**Results:** We demonstrate that in the prion infected cell lines, transient over-expression of the constitutively active mutant Rab7 reduces PrPSc propagation. Since prion infected cell cultures exhibit an increase in the cholesterol levels, we analysed how different functional mutants of Rab7 regulate cholesterol levels and observed that over-expression of the constitutively active Q67L mutant reduces the cholesterol level with respect to Rab7-WT mutant in 22LN2a cells. Over-expression of Rab7-WT did neither affect PrPSc levels, nor cholesterol content in those cells. This indicates that activation of Rab7 by GDP/GTP exchange might be impaired in 22LN2a cells. Preliminary results of a comparative analysis of Rab7-GTP in N2a and 22LN2a point at a reduction of Rab7-GTP upon prion infection. Since Rab7 is required for LDL transport, LDL trafficking is analysed to define a link between aberrant vesicle trafficking and enhanced cholesterol synthesis.

**Conclusion:** Through this study, we shed light on the underlying mechanisms and effects of impaired membrane association of Rab7 in prion infection. These effects include deterioration of cellular processes such as vesicle trafficking and cholesterol metabolism, hence favouring PrPSc propagation.

## 59. The role of glial cells in BSE pathology: a histomorphological and immunohistochemical investigation

Christine Fastª, Kristina Santiago-Mateoᵇ, Rakhi Katochᵇ, Martin H. Groschupª, Ute Zieglerª, Stefanie Czubᵇ

ªFriedrich-Loeffler Institut, Isle of Riems, Germany; ᵇCanadian Food Inspection Agency, Lethbridge, Alberta, Canada

CONTACT Christine.Fast@fli.de

## ABSTRACT

**Background/Introduction:** Bovine spongiform encephalopathy (BSE) is a fatal neurological disease of cattle associated with the accumulation of an altered form of the cellular prion protein, termed pathological prion protein (PrP<sup>BSE</sup>) The pathomechanisms linking abnormal protein accumulation with the typical severe neurological alterations in BSE are less than clear. Here we investigated the

role of glia cells in the pathology of BSE infected cattle, specifically if and to which degree the histopathological changes are glia mediated and therefore due to the host response and not to the BSE agent itself.

**Material and Methods:** We examined 29 experimentally BSE infected cattle at preclinical (no PrP<sup>BSE</sup> in the brain stem), late preclinical (no clinical signs, first traces of PrP<sup>BSE</sup> in the brain stem), and clinical (clinical signs and distinct accumulation of PrP<sup>BSE</sup> in the brain stem) stages of the disease. The following defined regions of the CNS were examined: Obex, Red Nucleus, Cerebellum, Parietal Cerebrum, Septal Nucleus, and Hippocampus and both histopathological lesions and PrP<sup>BSE</sup> accumulation were determined. Additionally, antibodies Iba-1, CX3CR1, and GFAP were used to visualize and identify glial involvement within the context of the disease. In addition, four unchallenged animals of the same age groups were included as negative controls.

**Results:** PrP<sup>BSE</sup> is found only in neurons and microglia. Different analytic approaches showed that the most consistent changes in glial numbers and phenotypes were detected in the Obex, Red Nucleus, and Hippocampus. However, only in clinical affected cattle, we observed with all antibodies a mild diffuse increase in glial reactivity, indicating a gliosis, as compared to controls, preclinical, and late preclinical animals. Additionally, different glial reaction patterns were almost exclusively detected in clinical animals. This includes a hyperplastic and in parts irregular appearance of the glial network, an accumulation of clumsy cells with shortened processes and rounded cell bodies, a diffusely distributed coarse granular staining and occasionally perineuronal cluster of glial cells.

**Conclusion:** Apart from neurons, we identified microglia as the only other PrP<sup>BSE</sup> accumulating cell population; computerized assessment of IBA1, CX3CR1, and GFAP antibodies are ongoing. The preliminary results presented here indicate that the activation of a glial response is a rather late event, most probably associated with the accumulation of PrP<sup>BSE</sup>.

## 60. Caribou Prnp polymorphism distribution in Canada and its impact on CWD pathogenesis

Maria I. Arifinª, Samia Hannaouiª, Yuan-Hung Huangª, Gordon Mitchellᵇ, Antanas Staskeviciusᵇ, Lech Kaczmarczykᶜ,ᵈ, Walker Jacksonᶜ,ᵈ and Sabine Gilchª

<footnote>ªDepartment of Ecosystem and Public Health, University of Calgary;</footnote>

<footnote>ᵇNational and OIE Reference Laboratory for Scrapie and CWD, Ottawa Laboratory Fallowfield, Canadian Food Inspection Agency;</footnote>

<footnote>ᶜWallenberg Center for Molecular Medicine, Department of Clinical</footnote>
<figure><img src="image_38.png" /></figure>

and Experimental Medicine, Linköping University; dGerman Center for Neurodegenerative Disease (DZNE), Bonn

CONTACT Maria I. Arifin maria.arifin@ucalgary.ca

## ABSTRACT

Wild reindeer in Norway were recently diagnosed with chronic wasting disease (CWD) [1]. Prions from CWD-infected deer were also transmissible to other reindeer in experimental settings [2,3]. Although CWD has not yet been reported in wild reindeer (caribou) in Canada, these studies show that they are at risk of infection. The wild-type reindeer, homozygous for serine at prion protein (PrP) residue 138 (138SS), developed clinical disease upon oral inoculation. Animals carrying at least one asparagine allele (138SN, 138NN) accumulated prions only in the periphery. However, both genotypes were susceptible to intracerebral prion inoculation [2,3]. Fallow deer are wild-type 138NN and are resistant to peripheral but not intracerebral prion infection [4]. Thus, we hypothesize that the138N allele, present in caribou herds in Alberta [5], alters CWD pathogenesis by limiting prion transport from the periphery to the CNS. Our aim is to elucidate the mechanisms involved in this process.

We extracted DNA from ±800 caribou, sequenced the *Prnp* open reading frame and determined the 138N allele prevalence in Canadian caribou populations. Results show that the 138N allele was highly prevalent in the Chinchaga woodland caribou population in BC (64%). It was also higher in barren-ground (37%) than in other woodland caribou populations (26%). To analyse how the 138N allele affects CWD pathogenesis, we generated knock-in (KI) mice where the mouse PrP is replaced by wild-type 138SS or 138NN cervid PrP. The KIs were obtained by injecting CRISPR/Cas9-edited C57BL6 embryonic stem cells (Bruce4) into wild-type blastocysts, generating chimeras, and breeding progeny to homozygosity in a C57BL6 background. PrP expression was determined using western blotting and qPCR. Correct PrP post-translational modifications were confirmed by PNGase-F and Endo-H digestion. We will inoculate our KIs with CWD-positive material from the corresponding reindeer genotypes [1,2]. The CWD-infected reindeer material was characterized by real-time quaking-induced conversion (RT-QuIC) and its transmissibility to transgenic mice overexpressing elk PrP (TgElk). Attack rate in the TgElks were almost 100%, except for those inoculated with 138SN lymph node material. Western blotting confirmed the presence of proteinase-K resistant PrP in all terminally ill mice. Our goal is to analyse the susceptibility of KIs carrying the 138N allele to intracerebral and peripheral prion infection. We will also assess the efficiency of prion transport from the periphery to the CNS in

intraperitoneally inoculated KIs. Prion strain propagation within a host is highly dependent on its PrP genotype. This study will provide insight into how the 138N PrP specifically influences CWD propagation in caribou.

**KEYWORDS:** Chronic wasting disease; polymorphism; caribou; reindeer; cervid; transgenic mice; knock-in mice

## References

[1] Benestad, et al. Vet Res. 2016;47:1.

[2] Mitchell, et al. PLoS ONE. 2012;7(6):e39055.

[3] Moore, et al. Emerg Infect Dis 2016;22(12):2142–5.

[4] Rhyan, et al. J Wildl Dis. 2011;47(3):739–4.

[5] Cheng, et al. Prion. 2017;11(2):136–42.

# 61. Prion propagation in cultured astrocytes: a model for prion infection in astroglia

Waqas Tahirª, Basant A Abdulrahmanª, Dalia H Abdelazizª, Simrika Thapaª, Rupali Waliaª and Hermann M Schatzlª

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

CONTACT Waqas Tahir waqas.tahir1@ucalgary.ca

## ABSTRACT

**Background:** Neuronal cell lines permissive to prion infection are useful experimental tools for studying the molecular mechanisms underlying prion infection. Such cell models are widely used to find and characterize chemical compounds which have anti-prion potential. Unfortunately, findings from cell culture are not very predictive for *in vivo* efficacy of such compounds. One reason might be that other cell types in the brain propagate prions, and that their processing of prions might differ from that of neurons. Exactly how astrocytes propagate, clear and disseminate prions is not well studied, explained by the lack of astrocyte cell models permissive of stable prion infection.

**Objective:** We investigated the permissiveness of an immortalized and uncloned astrocyte cell line to prion propagation, in order to develop a persistent prion infection model of astrocytes.

**Material and Methods:** We quantified mRNA and cell surface levels of PrPC in astrocytes with qPCR and FACS analysis, respectively, and compared them to those of other permissive neuronal (N2a and CAD5) and non-neuronal (MEF) cell lines. Next, we inoculated immortalized astrocytes with three mouse-adapted scrapie prion strains (22L,
<figure><img src="image_42.png" /></figure>

and Experimental Medicine, Linköping University; dGerman Center for Neurodegenerative Disease (DZNE), Bonn

CONTACT Maria I. Arifin maria.arifin@ucalgary.ca

## ABSTRACT

Wild reindeer in Norway were recently diagnosed with chronic wasting disease (CWD) [1]. Prions from CWD-infected deer were also transmissible to other reindeer in experimental settings [2,3]. Although CWD has not yet been reported in wild reindeer (caribou) in Canada, these studies show that they are at risk of infection. The wild-type reindeer, homozygous for serine at prion protein (PrP) residue 138 (138SS), developed clinical disease upon oral inoculation. Animals carrying at least one asparagine allele (138SN, 138NN) accumulated prions only in the periphery. However, both genotypes were susceptible to intracerebral prion inoculation [2,3]. Fallow deer are wild-type 138NN and are resistant to peripheral but not intracerebral prion infection [4]. Thus, we hypothesize that the138N allele, present in caribou herds in Alberta [5], alters CWD pathogenesis by limiting prion transport from the periphery to the CNS. Our aim is to elucidate the mechanisms involved in this process.

We extracted DNA from ±800 caribou, sequenced the *Prnp* open reading frame and determined the 138N allele prevalence in Canadian caribou populations. Results show that the 138N allele was highly prevalent in the Chinchaga woodland caribou population in BC (64%). It was also higher in barren-ground (37%) than in other woodland caribou populations (26%). To analyse how the 138N allele affects CWD pathogenesis, we generated knock-in (KI) mice where the mouse PrP is replaced by wild-type 138SS or 138NN cervid PrP. The KIs were obtained by injecting CRISPR/Cas9-edited C57BL6 embryonic stem cells (Bruce4) into wild-type blastocysts, generating chimeras, and breeding progeny to homozygosity in a C57BL6 background. PrP expression was determined using western blotting and qPCR. Correct PrP post-translational modifications were confirmed by PNGase-F and Endo-H digestion. We will inoculate our KIs with CWD-positive material from the corresponding reindeer genotypes [1,2]. The CWD-infected reindeer material was characterized by real-time quaking-induced conversion (RT-QuIC) and its transmissibility to transgenic mice overexpressing elk PrP (TgElk). Attack rate in the TgElks were almost 100%, except for those inoculated with 138SN lymph node material. Western blotting confirmed the presence of proteinase-K resistant PrP in all terminally ill mice. Our goal is to analyse the susceptibility of KIs carrying the 138N allele to intracerebral and peripheral prion infection. We will also assess the efficiency of prion transport from the periphery to the CNS in

intraperitoneally inoculated KIs. Prion strain propagation within a host is highly dependent on its PrP genotype. This study will provide insight into how the 138N PrP specifically influences CWD propagation in caribou.

**KEYWORDS:** Chronic wasting disease; polymorphism; caribou; reindeer; cervid; transgenic mice; knock-in mice

## References

[1] Benestad, et al. Vet Res. 2016;47:1.

[2] Mitchell, et al. PLoS ONE. 2012;7(6):e39055.

[3] Moore, et al. Emerg Infect Dis 2016;22(12):2142–5.

[4] Rhyan, et al. J Wildl Dis. 2011;47(3):739–4.

[5] Cheng, et al. Prion. 2017;11(2):136–42.

# 61. Prion propagation in cultured astrocytes: a model for prion infection in astroglia

Waqas Tahirª, Basant A Abdulrahmanª, Dalia H Abdelazizª, Simrika Thapaª, Rupali Waliaª and Hermann M Schatzlª

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

CONTACT Waqas Tahir waqas.tahir1@ucalgary.ca

## ABSTRACT

**Background:** Neuronal cell lines permissive to prion infection are useful experimental tools for studying the molecular mechanisms underlying prion infection. Such cell models are widely used to find and characterize chemical compounds which have anti-prion potential. Unfortunately, findings from cell culture are not very predictive for *in vivo* efficacy of such compounds. One reason might be that other cell types in the brain propagate prions, and that their processing of prions might differ from that of neurons. Exactly how astrocytes propagate, clear and disseminate prions is not well studied, explained by the lack of astrocyte cell models permissive of stable prion infection.

**Objective:** We investigated the permissiveness of an immortalized and uncloned astrocyte cell line to prion propagation, in order to develop a persistent prion infection model of astrocytes.

**Material and Methods:** We quantified mRNA and cell surface levels of PrP<sup>C</sup> in astrocytes with qPCR and FACS analysis, respectively, and compared them to those of other permissive neuronal (N2a and CAD5) and non-neuronal (MEF) cell lines. Next, we inoculated immortalized astrocytes with three mouse-adapted scrapie prion strains (22L,
<figure><img src="image_39.png" /></figure>

RML and ME7), and cultured them continuously for multiple passages. Prion propagation was examined with immunoblotting, RT-QuIC, and immunofluorescence methods at every passage.

**Results:** Surface expression of $PrP^C$ was comparable between astrocytes and N2a and CAD5 cells, and was higher than in MEF cells. Astrocytes infected with 22L prions showed $PrP^{Sc}$ in immunoblot analysis throughout all tested passages. Similarly, immunofluorescence results showed that astrocytes were able to propagate 22L prions stably. Infection with Me7 prions was negative in immunoblot, with transient weak signals in immunofluorescence and RT-QuIC analysis. Although RML infection was negative in immunoblot until passage 6 p.i., RML-infected astrocytes had the strongest signal in RT-QuIC in all passages (p1-p6).

**Conclusions:** Our data show that this uncloned astrocyte cell line is permissive to stable prion infection, depending on the prion strain. Whereas infection with 22L prions resulted in the expected strong signals in immunoblot, RT-QuIC, and immunofluorescence analysis, infection with RML prions yielded a stable infection which was positive in RT-QuIC and mostly negative in immunoblot analysis. Obviously, RML and 22L prions were handled very differently by this cell line, with regard to prion conversion and PK resistance. This new cell model will be helpful for better understanding of the role of astrocytes in prion infection.

## 62. A novel ex vivo model for characterization of Alzheimer's disease strains

Hailey E. Pineau<sup>a,b</sup>, Grant Norman<sup>a,b</sup>, David Westaway<sup>a,b</sup> and Valerie L. Sim<sup>a,b</sup>

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

CONTACT Hailey E. Pineau pineau@ualberta.ca

### ABSTRACT

**Background:** Affecting >48 million people worldwide, Alzheimer's disease (AD) is the leading cause of dementia. Evidence suggests that both amyloid $\beta$ ($A\beta$) and tau, the proteins associated with neurodegeneration in AD, exist in many different conformations (strains), which may explain variation in pathology and phenotype between AD patients. Specifically, we are interested in whether strain differences underlie differences in rate of disease progression. Using biophysical techniques and prion-based slice culture assays, we hope to elucidate $A\beta$ properties and plaque morphology characteristic of typical and

rapidly progressive AD, and to develop a novel ex vivo model that can replicate strain features.

**Methods:** The organotypic slice culture assay (OSCA) is a mouse brain slice culture that can be infected with different strains of prions and undergoes pathology as seen *in vivo*. Because $A\beta$ and tau possess prion-like infectivity, we are adapting this technique to AD using coronal brain slice cultures from 5xFAD and CRND8 mice. These mice express human mutant amyloid precursor protein and develop plaques and other neuropathology by 2 and 3 months of age, respectively. Typical or rapidly progressive AD brain homogenate will be applied to these slice cultures to induce strain-specific aggregates. Using confocal microscopy, we will quantify plaque burden, morphology, localization, and adjacent pathology. ELISA will be done to quantify levels of $A\beta$40 and $A\beta$42 in the AD brain samples and slice culture. Guanidinium denaturation curves will allow for comparison of $A\beta$ conformational stability between typical and rapidly progressive AD brain samples. Additionally, real-time quaking induced conversion (RT-QuIC) will be performed to assess seeding ability and aggregation kinetics of the $A\beta$ in all of the AD samples.

**Results:** Confocal images of $A\beta$ plaques stained with thioflavin S and/or 6E10 in fresh and cultured slices will be compared, and the effects of exposure to rapidly progressive AD brain homogenate will be shown. ELISA results will demonstrate a positive correlation between disease duration and hippocampal levels of $A\beta$42 and RT-QuIC data on aggregation kinetics may also be presented.

**Conclusions:** An ex-vivo model of AD that can replicate strain features will facilitate investigation into AD pathogenesis and strain-specific treatments.

## 63. Alteration of prion strain emergence by non-host factors

Sara A. M. Holec and Jason C. Bartz

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

CONTACT Sara A. M. Holec SaraHolec@creighton.edu

### ABSTRACT

Prions can persist in the environment for extended periods of time after adsorption to surfaces including soils, feeding troughs, or fences. The adsorption of $PrP^{Sc}$ to surfaces is influenced by a several factors including the prion strain, species of origin, and surface
<figure><img src="image_43.png" /></figure>

RML and ME7), and cultured them continuously for multiple passages. Prion propagation was examined with immunoblotting, RT-QuIC, and immunofluorescence methods at every passage.

**Results:** Surface expression of PrP<sup>C</sup> was comparable between astrocytes and N2a and CAD5 cells, and was higher than in MEF cells. Astrocytes infected with 22L prions showed PrP<sup>Sc</sup> in immunoblot analysis throughout all tested passages. Similarly, immunofluorescence results showed that astrocytes were able to propagate 22L prions stably. Infection with Me7 prions was negative in immunoblot, with transient weak signals in immunofluorescence and RT-QuIC analysis. Although RML infection was negative in immunoblot until passage 6 p.i., RML-infected astrocytes had the strongest signal in RT-QuIC in all passages (p1-p6).

**Conclusions:** Our data show that this uncloned astrocyte cell line is permissive to stable prion infection, depending on the prion strain. Whereas infection with 22L prions resulted in the expected strong signals in immunoblot, RT-QuIC, and immunofluorescence analysis, infection with RML prions yielded a stable infection which was positive in RT-QuIC and mostly negative in immunoblot analysis. Obviously, RML and 22L prions were handled very differently by this cell line, with regard to prion conversion and PK resistance. This new cell model will be helpful for better understanding of the role of astrocytes in prion infection.

**62. A novel ex vivo model for characterization of Alzheimer's disease strains**

Hailey E. Pineau<sup>a,b</sup>, Grant Norman<sup>a,b</sup>, David Westaway<sup>a,b</sup> and Valerie L. Sim<sup>a,b</sup>

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

CONTACT Hailey E. Pineau 
pineau@ualberta.ca


**ABSTRACT**

**Background:** Affecting >48 million people worldwide, Alzheimer's disease (AD) is the leading cause of dementia. Evidence suggests that both amyloid β (Aβ) and tau, the proteins associated with neurodegeneration in AD, exist in many different conformations (strains), which may explain variation in pathology and phenotype between AD patients. Specifically, we are interested in whether strain differences underlie differences in rate of disease progression. Using biophysical techniques and prion-based slice culture assays, we hope to elucidate Aβ properties and plaque morphology characteristic of typical and

rapidly progressive AD, and to develop a novel *ex vivo* model that can replicate strain features.

**Methods:** The organotypic slice culture assay (OSCA) is a mouse brain slice culture that can be infected with different strains of prions and undergoes pathology as seen *in vivo*. Because Aβ and tau possess prion-like infectivity, we are adapting this technique to AD using coronal brain slice cultures from 5xFAD and CRND8 mice. These mice express human mutant amyloid precursor protein and develop plaques and other neuropathology by 2 and 3 months of age, respectively. Typical or rapidly progressive AD brain homogenate will be applied to these slice cultures to induce strain-specific aggregates. Using confocal microscopy, we will quantify plaque burden, morphology, localization, and adjacent pathology. ELISA will be done to quantify levels of Aβ40 and Aβ42 in the AD brain samples and slice culture. Guanidinium denaturation curves will allow for comparison of Aβ conformational stability between typical and rapidly progressive AD brain samples. Additionally, real-time quaking induced conversion (RT-QuIC) will be performed to assess seeding ability and aggregation kinetics of the Aβ in all of the AD samples.

**Results:** Confocal images of Aβ plaques stained with thioflavin S and/or 6E10 in fresh and cultured slices will be compared, and the effects of exposure to rapidly progressive AD brain homogenate will be shown. ELISA results will demonstrate a positive correlation between disease duration and hippocampal levels of Aβ42 and RT-QuIC data on aggregation kinetics may also be presented.

**Conclusions:** An *ex-vivo* model of AD that can replicate strain features will facilitate investigation into AD pathogenesis and strain-specific treatments.

**63. Alteration of prion strain emergence by non-host factors**

Sara A. M. Holec and Jason C. Bartz

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

CONTACT Sara A. M. Holec 
SaraHolec@creighton.edu


**ABSTRACT**

Prions can persist in the environment for extended periods of time after adsorption to surfaces including soils, feeding troughs, or fences. The adsorption of PrP<sup>Sc</sup> to surfaces is influenced by a several factors including the prion strain, species of origin, and surface
<figure><img src="image_40.png" /></figure>

type. Attachment to surfaces can alter $PrP^{Sc}$ properties. The cumulative effect of these factors can result in strain- and soil-specific differences in prion adsorption, infectivity, and response to inactivation. Environmental processes such as wetting and drying or freezing and thawing can decrease $PrP^{Sc}$ conversion efficiency and enhance $PrP^{Sc}$ degradation. Little is known about how strain-specific incomplete inactivation of surface bound $PrP^{Sc}$ affects transmission and strain emergence. To examine this biologically relevant mode of prion transmission, we investigated the effect of surface binding and environmental forces on the emergence of a strain from a mixture.

To investigate the effects of environmental forces on strain emergence, we tested multiple parameters including proteolytic digestion, surface adsorption, and repeated cycles of wetting and drying. Extensive proteinase K (PK) digestion of HY and DY $PrP^{Sc}$ resulted in earlier emergence of HY $PrP^{Sc}$ in protein misfolding cyclic amplification strain interference (PMCAsi) compared to undigested controls. This result confirmed the proof of principle that selective alteration of the starting ratios of conversion competent HY and DY $PrP^{Sc}$ can alter strain emergence. We then further investigated if environmentally relevant factors such as surface binding and weathering could have a similar effect. Adsorption of HY and DY $PrP^{Sc}$ to silty clay loam (SCL) reduces conversion efficiency that can be strain-dependent; however, adsorption of HY and DY $PrP^{Sc}$ to SCL, both individually and together, did not alter strain emergence compared to unbound control PMCAsi reactions. Similarly, repeated cycles of wetting and drying of HY and DY $PrP^{Sc}$ bound to SCL did not alter the emergence of HY $PrP^{Sc}$. This data suggests that prion strain interference can occur when bound to surfaces. Since it is likely that surface bound $PrP^{Sc}$ is responsible for a significant portion of prion transmission under natural conditions, this is a highly significant finding. Under the limited conditions tested, we found that surface binding and weathering did not alter the emergence of a HY $PrP^{Sc}$.

## 64. Investigation of the role of lamin A-induced molecular ageing in tauopathy

Zhuang (George) Han AND David Westaway

Centre for Prions and Protein Folding Diseases, Department of Biochemistry, Faculty of Medicine and Dentistry, University of Alberta

CONTACT Zhuang (George) Han zzhan@ualberta.ca

### ABSTRACT

Tauopathies are a diverse group of age-related neurodegenerative diseases characterised by filamentous Tau

lesions in brain [1]. Alzheimer's disease, frontotemporal dementia and progressive supranuclear palsy are examples of tauopathies that have presented a tremendous challenge to health care systems worldwide [2-4]. Tau is a microtubule-associated protein predominantly expressed in axons of neurons. In disease states, it becomes hyperphosphorylated and migrates to the cell body, forming aggregates that are toxic to neurons [1,5]. Certain tauopathies may associate with impaired lamin-enriched nuclear lamina and thus are also considered laminopathies, at least in the experimental context of *Drosophila melanogaster* [6,7].

Hutchinson Gilford progeria syndrome (HGPS), an autosomal dominant disorder of premature ageing, associates with a truncated form of lamin A (termed 'progerin') [8,9]. Neurons in the brain mainly produce abundant lamin C but little lamin A, a consequence of prelamin A mRNA degradation by a brain-specific microRNA [10]. Although this reasonably explains the absence of neurological symptoms in lamin A-induced accelerated ageing, it remains unclear whether progerin - if it were expressed in neurons - would enhance pathological Tau-induced neurotoxicity in brain, given that overexpression of progerin can be toxic to enteric nerves and stem cell-derived neurons [11,12]. To address this question, we are using the HGPS progeric mutation of lamin A to accelerate cellular ageing in petri-dish cultures; we hypothesise that when the expression level of aggregation-prone Tau is low (near-physiological level), lamin A- induced cellular changes might amplify or alter its pathogenic potential.

Progeric and wildtype forms of lamin A were transfected into an HEK293 Tau RD-YFP reporter cell line [13] with subcellular localization of progerin and perturbation of the nuclear lamina assessed by immunohistochemistry. Following transduction of different classes of Tau fibril derived from transgenic P301L mice and human P301L patients, morphologies of Tau aggregates in HEK reporter cells with and without lamin A expression are determined by fluorescence microscopy. Aggregated Tau is then harvested, digested with trypsin and analysed with capillary westerns and folding state-dependent immunoassays. Based upon results from tauopathy cell culture model, the HGPS progeric mutation may be applied to animal studies to augment the use of chronological ageing as a parameter in studies of Tau-mediated pathogenesis.

**KEYWORDS:** Tauopathy; laminopathy; progeria syndrome; ageing; neurodegenerative disease

### References

[1] Lee VM, Goedert M, Trojanowski JQ. Neurodegenerative tauopathies. Annu Rev Neurosci. 2001;24:1121-59.
<figure><img src="image_44.png" /></figure>

type. Attachment to surfaces can alter $PrP^{Sc}$ properties. The cumulative effect of these factors can result in strain- and soil-specific differences in prion adsorption, infectivity, and response to inactivation. Environmental processes such as wetting and drying or freezing and thawing can decrease $PrP^{Sc}$ conversion efficiency and enhance $PrP^{Sc}$ degradation. Little is known about how strain-specific incomplete inactivation of surface bound $PrP^{Sc}$ affects transmission and strain emergence. To examine this biologically relevant mode of prion transmission, we investigated the effect of surface binding and environmental forces on the emergence of a strain from a mixture.

To investigate the effects of environmental forces on strain emergence, we tested multiple parameters including proteolytic digestion, surface adsorption, and repeated cycles of wetting and drying. Extensive proteinase K (PK) digestion of HY and DY $PrP^{Sc}$ resulted in earlier emergence of HY $PrP^{Sc}$ in protein misfolding cyclic amplification strain interference (PMCAsi) compared to undigested controls. This result confirmed the proof of principle that selective alteration of the starting ratios of conversion competent HY and DY $PrP^{Sc}$ can alter strain emergence. We then further investigated if environmentally relevant factors such as surface binding and weathering could have a similar effect. Adsorption of HY and DY $PrP^{Sc}$ to silty clay loam (SCL) reduces conversion efficiency that can be strain-dependent; however, adsorption of HY and DY $PrP^{Sc}$ to SCL, both individually and together, did not alter strain emergence compared to unbound control PMCAsi reactions. Similarly, repeated cycles of wetting and drying of HY and DY $PrP^{Sc}$ bound to SCL did not alter the emergence of HY $PrP^{Sc}$. This data suggests that prion strain interference can occur when bound to surfaces. Since it is likely that surface bound $PrP^{Sc}$ is responsible for a significant portion of prion transmission under natural conditions, this is a highly significant finding. Under the limited conditions tested, we found that surface binding and weathering did not alter the emergence of a HY $PrP^{Sc}$.

**64. Investigation of the role of lamin A-induced molecular ageing in tauopathy**

Zhuang (George) Han AND David Westaway

Centre for Prions and Protein Folding Diseases, Department of Biochemistry, Faculty of Medicine and Dentistry, University of Alberta

CONTACT Zhuang (George) Han zzhan@ualberta.ca

**ABSTRACT**

Tauopathies are a diverse group of age-related neurodegenerative diseases characterised by filamentous Tau

lesions in brain [1]. Alzheimer's disease, frontotemporal dementia and progressive supranuclear palsy are examples of tauopathies that have presented a tremendous challenge to health care systems worldwide [2-4]. Tau is a microtubule-associated protein predominantly expressed in axons of neurons. In disease states, it becomes hyperphosphorylated and migrates to the cell body, forming aggregates that are toxic to neurons [1,5]. Certain tauopathies may associate with impaired lamin-enriched nuclear lamina and thus are also considered laminopathies, at least in the experimental context of *Drosophila melanogaster* [6,7].

Hutchinson Gilford progeria syndrome (HGPS), an autosomal dominant disorder of premature ageing, associates with a truncated form of lamin A (termed 'progerin') [8,9]. Neurons in the brain mainly produce abundant lamin C but little lamin A, a consequence of prelamin A mRNA degradation by a brain-specific microRNA [10]. Although this reasonably explains the absence of neurological symptoms in lamin A-induced accelerated ageing, it remains unclear whether progerin - if it were expressed in neurons - would enhance pathological Tau-induced neurotoxicity in brain, given that overexpression of progerin can be toxic to enteric nerves and stem cell-derived neurons [11,12]. To address this question, we are using the HGPS progeric mutation of lamin A to accelerate cellular ageing in petri-dish cultures; we hypothesise that when the expression level of aggregation-prone Tau is low (near-physiological level), lamin A- induced cellular changes might amplify or alter its pathogenic potential.

Progeric and wildtype forms of lamin A were transfected into an HEK293 Tau RD-YFP reporter cell line [13] with subcellular localization of progerin and perturbation of the nuclear lamina assessed by immunohistochemistry. Following transduction of different classes of Tau fibril derived from transgenic P301L mice and human P301L patients, morphologies of Tau aggregates in HEK reporter cells with and without lamin A expression are determined by fluorescence microscopy. Aggregated Tau is then harvested, digested with trypsin and analysed with capillary westerns and folding state-dependent immunosays. Based upon results from tauopathy cell culture model, the HGPS progeric mutation may be applied to animal studies to augment the use of chronological ageing as a parameter in studies of Tau-mediated pathogenesis.

**KEYWORDS:** Tauopathy; laminopathy; progeria syndrome; ageing; neurodegenerative disease

**References**

[1] Lee VM, Goedert M, Trojanowski JQ. Neurodegenerative tauopathies. Annu Rev Neurosci. 2001;24:1121-59.
<figure><img src="image_41.png" /></figure>

[2] Nath U, Ben-Shlomo Y, Thomson RG, et al. The prevalence of progressive supranuclear palsy (Steele-Richardson-Olszewski syndrome) in the UK. Brain 2001;124(Pt 7):1438-49.

[3] Plassman BL, Langa KM, Fisher GG, et al. Prevalence of dementia in the United States: the aging, demographics, and memory study. Neuroepidemiology. 2007;29:125-132.

[4] Onyike CU, Diehl-Schmid J. The epidemiology of frontotemporal dementia. Int Rev Psychiatry. 2013;25(2):130-137.

[5] Mietelska-Porowska A, Wasik U, Goras M, et al. Tau protein modifications and interactions: their role in function and dysfunction. Int. J. Mol. Sci. 2014;15:4671-4713.

[6] Frost, et al. Alzheimer's disease: an acquired neurodegenerative laminopathy. Nucleus. 2016;7(3):275-283.

[7] Frost, Bardai FH, Feany MB, et al. Lamin dysfunction mediates neurodegeneration in tauopathies. Current Biol. 2016;26:129-136.

[8] Pollex RL, Hegele RA. Hutchinson-Gilford progeria syndrome. Clin Genet. 2004;66(5):375-81.

[9] Schreiber KH, Kennedy, BK. When lamins go bad: nuclear structure and disease. Cell 2013. 152(6):1365-75.

[10] Jung HJ, Coffinier C, Choe Y, et al. Regulation of prelamin A but not lamin C by miR-9, a brain-specific microRNA. Proc. Natl Acad. Sci. 2012;109:E423-E431.

[11] Yang SH, Procaccia S, Jung H-J. et al. Mice that express farnesylated versions of prelamin A in neurons develop achalasia. Human Mol. Genet. 2015;24(10):2826-2840.

[12] Miller JD, Ganat YM, Kishinevsky S, et al. Human iPSC-based modeling of late-onset disease via progerin-induced aging. Cell Stem Cell. 2013;13:691-705.

[13] Sanders DW, Kaufman SK, DeVos SL, et al. Distinct tau prion strains propagate in cells and mice and define different tauopathies. Neuron 2014;82:1271-1288.

## 65. vCJD strain is consistent in individuals of two PRNP codon 129 genotypes

Abigail B. Diackª, Aileen Boyleª, Chris Plinstonª, Diane Ritchieᵇ, Emma Huntª, Matthew Bishopᵇ, Robert G. Willᵇ and Jean C. Mansonᶜ

**CONTACT** Abigail B. Diack abigail.diack@roslin.ed.ac.uk

## ABSTRACT

**Background/Introduction:** The identification of a clinical case of vCJD in a PRNP 129 heterozygous individual in 2016 raised concerns over whether 129MV individuals can transmit the vCJD infection and whether the 129MV genotype could give rise to a

novel strain of vCJD; both of which have significant public health concerns. To date, of the 178 definite/ probable cases of vCJD, only one has been of the 129MV genotype however evidence of abnormal PrP has been observed in two asymptomatic cases of vCJD. We have now undertaken the strain characterization of both a clinical case of vCJD and that of an asymptomatic case of vCJD in 129MV individuals.

**Methods:** CNS material from a 129MV clinical case of vCJD [1] and spleen material from an asymptomatic 129MV individual [2] were inoculated into panels of wildtype mice. Serial passage of the spleen material from the asymptomatic 129MV individual was then performed by inoculating brain homogenate from a TSE positive mouse from each mouse strain into the wildtype mouse panel. This was then repeated for a second mouse passage. A combination of clinical signs, neuropathology (TSE vacuolation and PrP deposition) and biochemical analysis were carried out for each transmission and compared against 129MM and BSE transmission data.

**Results:** Primary passage of the clinical case of 129MV vCJD (CNS) in wildtype mice gave rise to pathologically confirmed (TSE vacuolation and PrP deposition) clinical disease. Differences in transmission data were observed at the primary and first subpassage of the asymptomatic 129MV case (spleen) compared to 129MM transmissions. These differences included low attack rates with no or little TSE vacuolation at primary passage and changes in incubation periods and rankings. These differences were resolved upon further passage giving rise to strain characteristics consistent with 129MM vCJD and BSE transmission data.

**Conclusions:** These studies comprise the first strain characterization of vCJD in 129MV individuals. Upon primary passage of the clinical case of 129MV vCJD transmission characteristics resemble that of the 129MM vCJD and BSE strain. While some differences were observed at primary and subpassage of the asymptomatic case, these resolved by the second subpassage and strain characteristics are totally consistent with 129MM vCJD. We demonstrate that vCJD strain properties are not affected by transmission through individuals with the 129MV genotype and thus no alteration in virulence should be associated with different host genotype.

## References

[1] Mok T, Jaunmuktane Z, Joiner S, et al. Variant Creutzfeldt-Jakob disease in a patient with heterozygosity at PRNP codon 129. N Engl J Med. 2017;376(3):292-4.

<footnote>ªThe Roslin Institute and R(D)SVS, University of Edinburgh, Easter Bush, UK; ᵇNational CJD Research & Surveillance Unit, Center for Clinical Brain Sciences, University of Edinburgh, Edinburgh, UK; ᶜUniversity of Edinburgh, Edinburgh, UK</footnote>
<figure><img src="image_45.png" /></figure>

[2] Nath U, Ben-Shlomo Y, Thomson RG, et al. The prevalence of progressive supranuclear palsy (Steele-Richardson-Olszewski syndrome) in the UK. Brain 2001;124(Pt 7):1438-49.

[3] Plassman BL, Langa KM, Fisher GG, et al. Prevalence of dementia in the United States: the aging, demographics, and memory study. Neuroepidemiology. 2007;29:125-132.

[4] Onyike CU, Diehl-Schmid J. The epidemiology of frontotemporal dementia. Int Rev Psychiatry. 2013;25(2):130-137.

[5] Mietelska-Porowska A, Wasik U, Goras M, et al. Tau protein modifications and interactions: their role in function and dysfunction. Int. J. Mol. Sci. 2014;15:4671-4713.

[6] Frost, et al. Alzheimer's disease: an acquired neurodegenerative laminopathy. Nucleus. 2016;7(3):275-283.

[7] Frost, Bardai FH, Feany MB, et al. Lamin dysfunction mediates neurodegeneration in tauopathies. Current Biol. 2016;26:129-136.

[8] Pollex RL, Hegele RA. Hutchinson-Gilford progeria syndrome. Clin Genet. 2004;66(5):375-81.

[9] Schreiber KH, Kennedy, BK. When lamins go bad: nuclear structure and disease. Cell 2013. 152(6):1365-75.

[10] Jung HJ, Coffinier C, Choe Y, et al. Regulation of prelamin A but not lamin C by miR-9, a brain-specific microRNA. Proc. Natl Acad. Sci. 2012;109:E423-E431.

[11] Yang SH, Procaccia S, Jung H-J. et al. Mice that express farnesylated versions of prelamin A in neurons develop achalasia. Human Mol. Genet. 2015;24(10):2826-2840.

[12] Miller JD, Ganat YM, Kishinevsky S, et al. Human iPSC-based modeling of late-onset disease via progerin-induced aging. Cell Stem Cell. 2013;13:691-705.

[13] Sanders DW, Kaufman SK, DeVos SL, et al. Distinct tau prion strains propagate in cells and mice and define different tauopathies. Neuron 2014;82:1271-1288.

**65. vCJD strain is consistent in individuals of two PRNP codon 129 genotypes**

Abigail B. Diackª, Aileen Boyleª, Chris Plinstonª, Diane Ritchieᵇ, Emma Huntª, Matthew Bishopᵇ, Robert G. Willᵇ and Jean C. Mansonᶜ

CONTACT Abigail B. Diack abigail.diack@roslin.ed.ac.uk

ABSTRACT

**Background/Introduction:** The identification of a clinical case of vCJD in a PRNP 129 heterozygous individual in 2016 raised concerns over whether 129MV individuals can transmit the vCJD infection and whether the 129MV genotype could give rise to a

novel strain of vCJD; both of which have significant public health concerns. To date, of the 178 definite/ probable cases of vCJD, only one has been of the 129MV genotype however evidence of abnormal PrP has been observed in two asymptomatic cases of vCJD. We have now undertaken the strain characterization of both a clinical case of vCJD and that of an asymptomatic case of vCJD in 129MV individuals.

**Methods:** CNS material from a 129MV clinical case of vCJD [1] and spleen material from an asymptomatic 129MV individual [2] were inoculated into panels of wildtype mice. Serial passage of the spleen material from the asymptomatic 129MV individual was then performed by inoculating brain homogenate from a TSE positive mouse from each mouse strain into the wildtype mouse panel. This was then repeated for a second mouse passage. A combination of clinical signs, neuropathology (TSE vacuolation and PrP deposition) and biochemical analysis were carried out for each transmission and compared against 129MM and BSE transmission data.

**Results:** Primary passage of the clinical case of 129MV vCJD (CNS) in wildtype mice gave rise to pathologically confirmed (TSE vacuolation and PrP deposition) clinical disease. Differences in transmission data were observed at the primary and first subpassage of the asymptomatic 129MV case (spleen) compared to 129MM transmissions. These differences included low attack rates with no or little TSE vacuolation at primary passage and changes in incubation periods and rankings. These differences were resolved upon further passage giving rise to strain characteristics consistent with 129MM vCJD and BSE transmission data.

**Conclusions:** These studies comprise the first strain characterization of vCJD in 129MV individuals. Upon primary passage of the clinical case of 129MV vCJD transmission characteristics resemble that of the 129MM vCJD and BSE strain. While some differences were observed at primary and subpassage of the asymptomatic case, these resolved by the second subpassage and strain characteristics are totally consistent with 129MM vCJD. We demonstrate that vCJD strain properties are not affected by transmission through individuals with the 129MV genotype and thus no alteration in virulence should be associated with different host genotype.

References

[1] Mok T, Jaunmuktane Z, Joiner S, et al. Variant Creutzfeldt-Jakob disease in a patient with heterozygosity at PRNP codon 129. N Engl J Med. 2017;376(3):292-4.

<footnote>ªThe Roslin Institute and R(D)SVS, University of Edinburgh, Easter Bush, UK; ᵇNational CJD Research & Surveillance Unit, Center for Clinical Brain Sciences, University of Edinburgh, Edinburgh, UK; ᶜUniversity of Edinburgh, Edinburgh, UK</footnote>
<figure><img src="image_42.png" /></figure>

[2] Peden AH, Head MW, Ritchie DL, et al. Mok TPreclinical vCJD after blood transfusion in a PRNP codon 129 heterozygous patient. Lancet. 2004;364 (9433):527-9.

## 66. Scrapie strain typing of brain and lymph node-derived isolates in ovinized models reveals mixture of substrains with distinct pathobiological properties

Tomás Barrioª, Hicham Filaliª, Alicia Oteroª, Jessica Sheleby-Elíasª, Belén Marínª, Enric Vidalᵇ, Juan María Torresᶜ, Martin Groschupᵈ, Olivier Andréolettiᵉ, Juan José Badiolaª and Rosa Boleaª

ªCentro de Encefalopatías y Enfermedades Transmisibles Emergentes, Facultad de Veterinaria, Instituto Agroalimentario de Aragón – IA2, Universidad de Zaragoza – CITA, Zaragoza, Spain; ᵇPriocat Laboratory, Centre de Recerca en Sanitat Animal (CReSA), UAB-IRTA, Universitat Autònoma de Barcelona (UAB), Barcelona, Spain; ᶜCentro de Investigación en Sanidad Animal, CISAINIA, Madrid, Spain; ᵈInstitute of Novel and Emerging Infectious Diseases, Friedrich-Loeffler-Institute, Greifswald-Isle of Riems, Germany; ᵉUMR INRA ENVT 1225- IHAP, École Nationale Vétérinaire de Toulouse, Toulouse, France

CONTACT Rosa Bolea rbolea@unizar.es

### ABSTRACT

Phenotypic variability has been observed in several prion diseases, including human TSEs and scrapie of sheep of goats. This heterogeneity has been associated with distinct prion strains. The existence of strains in prion biology can be accommodated within the protein-only hypothesis by supporting the view that several conformational variants of $PrP^{Sc}$ exist, which encode distinct pathobiological properties. Within this framework, the conformational selection model proposes that a given amino acid sequence for $PrP^{C}$ allows a limited portfolio of $PrP^{Sc}$ conformations, such that the degree of overlapping between host and donor Prnp sequences determines the capability of the isolate to cause disease.

Transmission barrier occurs upon heterologous interaction between incompatible PrP sequences, giving rise to a variety of new prion conformers. Therefore, transgenic models expressing homologous $PrP^{C}$ are crucial to faithfully study the actual variety of prion strains. Ovinized models show enhanced susceptibility to infection with scrapie prions and have been employed to characterize strains in natural sheep isolates.

In the present study, we used the transgenic murine lines TgShp and Tg338, which express ovine $PrP^{C}$ ARQ and VRQ, respectively, to bioassay 22 sheep scrapie isolates from distinct outbreaks within the Spain-France-Andorra transboundary territory. Animals were intracerebrally inoculated and survival periods, lesion profiles, $PrP^{Sc}$ distribution, and glycosylation patterns were studied.

In all cases but one, Western blot from sheep tissues disclosed glycosylation patterns compatible with a low molecular weight (Mw) scrapie strain (~19 kDa). Only Inoculum 8L showed $PrP^{res}$ of higher Mw (~21 kDa).

On bioassay, all inocula caused on second-passage TgShp mice similar survival periods together with high attack rates. While most TgShp mice accumulated 19-kDa $PrP^{res}$ in their spinal cords, a number of isolates (including inoculum 8L) triggered deposition of the 21-kDa isoform.

In Tg338 mice, a majority of isolates induced survival times similar to those seen in TgShp, together with high attack rates and 19-kDa $PrP^{res}$ in the spinal cord. However, a group of isolates showed different features consisting in very long survival periods with low attack rates and presence of 21-kDa $PrP^{res}$. Additionally, these animals showed lower vacuolization scores in all evaluated brain areas and occasional presence of amyloid plaques.

These results suggest that some scrapie isolates contain mixtures of substrains that are resolved distinctly in different transgenic lines. A number of isolates seemed to comprise a major 19-kDa component and a minor 21-kDa component that is specifically amplified by the TgShp, but not the Tg338 line. In contrast, some other isolates contained low titers of a 21-kDa, low pathogenicity isoform that seemed to interfere with the propagation of the major 19-kDa substrain exclusively in Tg338 thanks to its amyloidogenic properties, that sequester neurotoxic PrP monomers delaying the onset of clinical signs and restraining the development of neuropathology.

## 67. Diagnostic accuracy of cerebrospinal fluid RT-QuIC in cases of suspected prion disease and the potential utility of using RT-QuIC for public health surveillance

Daniel D. Rhoadsª,ᵇ, Aleksandra Wronaᶜ, Aaron Foutzª, Curtis Tatsuokaª,ᵈ, Janis Blevinsª, Ryan A. Maddoxᵉ, Ermias D. Belayᵉ, Lawrence B. Schonbergerᵉ, Mark L. Cohenª,ᵇ,ᵇ,ғ and Brian S. Applebyª,ᵇ,ғ,g

ªNational Prion Disease Pathology Surveillance Center, Case Western Reserve University, Cleveland, OH, USA; ᵇDepartment of Pathology, Case Western Reserve University, Cleveland, OH, USA; ᶜYale University, New Haven, CT, USA; ᵈDepartment of Biostatistics, Case Western Reserve University, Cleveland, OH, USA; ᵉCenters for Disease Control and Prevention, Atlanta, GA, USA; ᵈDepartment of Neurology, Case Western Reserve University, Cleveland, OH, USA; ᵉDepartment of Psychiatry, Case Western Reserve University, Cleveland, OH, USA

CONTACT Brian S. Appleby bsa35@case.edu

### ABSTRACT

**Background:** The clinical heterogeneity, potential mimickers, and rapid progression make prion diseases
<figure><img src="image_46.png" /></figure>

[2] Peden AH, Head MW, Ritchie DL, et al. Mok TPreclinical vCJD after blood transfusion in a PRNP codon 129 heterozygous patient. Lancet. 2004;364 (9433):527-9.

## 66. Scrapie strain typing of brain and lymph node-derived isolates in ovinized models reveals mixture of substrains with distinct pathobiological properties

Tomás Barrioª, Hicham Filaliª, Alicia Oteroª, Jessica Sheleby-Elíasª, Belén Marínª, Enric Vidalᵇ, Juan María Torresᶜ, Martin Groschupᵈ, Olivier Andréolettiᵉ, Juan José Badiolaª and Rosa Boleaª

ªCentro de Encefalopatías y Enfermedades Transmisibles Emergentes, Facultad de Veterinaria, Instituto Agroalimentario de Aragón – IA2, Universidad de Zaragoza – CITA, Zaragoza, Spain; ᵇPriocat Laboratory, Centre de Recerca en Sanitat Animal (CReSA), UAB-IRTA, Universitat Autònoma de Barcelona (UAB), Barcelona, Spain; ᶜCentro de Investigación en Sanidad Animal, CISAINIA, Madrid, Spain; ᵈInstitute of Novel and Emerging Infectious Diseases, Friedrich-Loeffler-Institute, Greifswald-Isle of Riems, Germany; ᵉUMR INRA ENVT 1225- IHAP, École Nationale Vétérinaire de Toulouse, Toulouse, France

CONTACT Rosa Bolea rbolea@unizar.es

### ABSTRACT

Phenotypic variability has been observed in several prion diseases, including human TSEs and scrapie of sheep of goats. This heterogeneity has been associated with distinct prion strains. The existence of strains in prion biology can be accommodated within the protein-only hypothesis by supporting the view that several conformational variants of $PrP^{Sc}$ exist, which encode distinct pathobiological properties. Within this framework, the conformational selection model proposes that a given amino acid sequence for $PrP^{C}$ allows a limited portfolio of $PrP^{Sc}$ conformations, such that the degree of overlapping between host and donor Prnp sequences determines the capability of the isolate to cause disease.

Transmission barrier occurs upon heterologous interaction between incompatible PrP sequences, giving rise to a variety of new prion conformers. Therefore, transgenic models expressing homologous $PrP^{C}$ are crucial to faithfully study the actual variety of prion strains. Ovinized models show enhanced susceptibility to infection with scrapie prions and have been employed to characterize strains in natural sheep isolates.

In the present study, we used the transgenic murine lines TgShp and Tg338, which express ovine $PrP^{C}$ ARQ and VRQ, respectively, to bioassay 22 sheep scrapie isolates from distinct outbreaks within the Spain-France-Andorra transboundary territory. Animals were intracerebrally inoculated and survival periods, lesion profiles, $PrP^{Sc}$ distribution, and glycosylation patterns were studied.

In all cases but one, Western blot from sheep tissues disclosed glycosylation patterns compatible with a low molecular weight (Mw) scrapie strain (~19 kDa). Only Inoculum 8L showed $PrP^{res}$ of higher Mw (~21 kDa).

On bioassay, all inocula caused on second-passage TgShp mice similar survival periods together with high attack rates. While most TgShp mice accumulated 19-kDa $PrP^{res}$ in their spinal cords, a number of isolates (including inoculum 8L) triggered deposition of the 21-kDa isoform.

In Tg338 mice, a majority of isolates induced survival times similar to those seen in TgShp, together with high attack rates and 19-kDa $PrP^{res}$ in the spinal cord. However, a group of isolates showed different features consisting in very long survival periods with low attack rates and presence of 21-kDa $PrP^{res}$. Additionally, these animals showed lower vacuolization scores in all evaluated brain areas and occasional presence of amyloid plaques.

These results suggest that some scrapie isolates contain mixtures of substrains that are resolved distinctly in different transgenic lines. A number of isolates seemed to comprise a major 19-kDa component and a minor 21-kDa component that is specifically amplified by the TgShp, but not the Tg338 line. In contrast, some other isolates contained low titers of a 21-kDa, low pathogenicity isoform that seemed to interfere with the propagation of the major 19-kDa substrain exclusively in Tg338 thanks to its amyloidogenic properties, that sequester neurotoxic PrP monomers delaying the onset of clinical signs and restraining the development of neuropathology.

## 67. Diagnostic accuracy of cerebrospinal fluid RT-QuIC in cases of suspected prion disease and the potential utility of using RT-QuIC for public health surveillance

Daniel D. Rhoadsª,ᵇ, Aleksandra Wronaᶜ, Aaron Foutzª, Curtis Tatsuokaª,ᵈ, Janis Blevinsª, Ryan A. Maddoxᵉ, Ermias D. Belayᵉ, Lawrence B. Schonbergerᵉ, Mark L. Cohenª,ᵇ,ᵇ,ғ and Brian S. Applebyª,ᵇ,ғ,г

ªNational Prion Disease Pathology Surveillance Center, Case Western Reserve University, Cleveland, OH, USA; ᵇDepartment of Pathology, Case Western Reserve University, Cleveland, OH, USA; ᶜYale University, New Haven, CT, USA; ᵈDepartment of Biostatistics, Case Western Reserve University, Cleveland, OH, USA; ᵉCenters for Disease Control and Prevention, Atlanta, GA, USA; ᵈDepartment of Neurology, Case Western Reserve University, Cleveland, OH, USA; ᵉDepartment of Psychiatry, Case Western Reserve University, Cleveland, OH, USA

CONTACT Brian S. Appleby bsa35@case.edu

### ABSTRACT

**Background:** The clinical heterogeneity, potential mimickers, and rapid progression make prion diseases
<figure><img src="image_43.png" /></figure>

difficult to diagnose. The only way to definitely diagnose prion disease is via neuropathologic examination, but reliable antemortem diagnoses can be made, especially with the advent of real time quaking induced conversion (RT-QuIC) to detect prions. Individuals with a neuropsychiatric disorder and positive RT-QuIC are considered to have probable sporadic Creutzfeldt-Jakob disease (sCJD) using current diagnostic criteria.

**Materials and Methods:** Demographic and laboratory data were examined for cerebrospinal fluid (CSF) specimens submitted to the National Prion Disease Pathology Surveillance Center (NPDPSC) from May 2015 to April 2018. In cases with multiple specimen submission, only the first specimen received was included in these analyses. Second generation RT-QuIC using truncated hamster prion protein substrate was performed on all specimens. Neuropathologic examination at autopsy was used as the gold standard comparator. Autopsy is biased in favour of cases where the diagnosis is uncertain at death, namely when false positive or false negative RT-QuIC results are suspected. Autopsy tissue received through October 2018 was included in the analyses.

**Results:** The NPDPSC received CSF specimens from 10,498 living patients with suspected prion disease, and 567 (5.4%) subsequently underwent neuropathologic examination by the NPDSPC. Overall, 1,103 (10.5%) CSF specimens submitted to the NPDPSC were RT-QuIC positive. At autopsy, 497 (87.7%) cases were positive for prion disease, including 437 (77.4%) with definitive sCJD. Overall CSF RT-QuIC diagnostic sensitivity was 90.3% (449/497) and specificity was 98.5% (69/70). Sensitivity was highest in sCJD (92.9%; 406/437) compared to genetic prion diseases (82%; 31/38). RT-QuIC was negative in all cases of sporadic fatal insomnia (sFI, *n* = 5), sCJD VV1 (*n* = 3), and fatal familial insomnia (FFI, *n* = 4). Younger subjects were more likely to have false negative RT-QuIC results. One false positive RT-QuIC test occurred in a case of multifactorial dementia (Alzheimer's disease and vascular).

**Conclusions:** We present analyses of a large cohort of CSF specimens and autopsy tissue submitted to the NPDPSC that includes high variability in demographics and prion disease subtypes. RT-QuIC demonstrated its highest sensitivity when testing CSF from older individuals and individuals with common forms of sCJD. RT-QuIC demonstrated poor sensitivity in detecting some uncommon prion disease subtypes (sFI, sCJD VV1, and FFI). The high specificity of RT-QuIC suggests that counting individuals who are positive for RT-QuIC in public health surveillance case numbers of probable/definitive prion disease, and antemortem CSF testing may foster improved prion disease surveillance.

## 68. Bioassays reveal Aβ and tau prions decline with longevity in Alzheimer's disease

Carlo Condello<sup>a,b</sup>, Atsushi Aoyagi<sup>a,c</sup>, Jan Stöhr<sup>a,b,d</sup>, Weizhou Yue<sup>a</sup>, Brianna M. Rivera<sup>a</sup>, Joanne C. Lee<sup>a</sup>, Amanda L. Woerman<sup>a,b</sup>, Glenda Halliday<sup>e</sup>, Sjoerd van Duinen<sup>f</sup>, Martin Ingelsson<sup>g</sup>, Lars Lannfelt<sup>g</sup>, Caroline Graff<sup>h,i</sup>, Thomas D. Bird<sup>j,k</sup>, C. Dirk Keene<sup>l</sup>, William W. Seeley<sup>b,m</sup>, William F. DeGrado<sup>a,n</sup> and Stanley B. Prusiner<sup>a,b,o</sup>

<sup>a</sup>Institute for Neurodegenerative Diseases, Weill Institute for Neurosciences, University of California, San Francisco, CA, USA;
<sup>b</sup>Department of Neurology, Weill Institute for Neurosciences, University of California, San Francisco, CA, USA;
<sup>c</sup>Daiichi Sankyo Co., Ltd., Tokyo, Japan;
<sup>d</sup>AC Immune SA, Lausanne, Switzerland;
<sup>e</sup>School of Medical Science, University of New South Wales, Sydney, Australia;
<sup>f</sup>Leiden University Medical Center, Leiden, The Netherlands;
<sup>g</sup>Department of Public Health and Caring Services/Geriatrics, Uppsala University, Uppsala, Sweden;
<sup>h</sup>Department of Neurobiology, Care Sciences and Society, Karolinska Institute, Huddinge, Sweden;
<sup>i</sup>Department of Geriatric Medicine, Karolinska University Hospital, Stockholm, Sweden;
<sup>j</sup>Department of Medicine, Division of Medical Genetics, University of Washington, Seattle, WA, USA;
<sup>k</sup>Department of Neurology, University of Washington, Seattle, WA, USA;
<sup>l</sup>Department of Neuropathology, University of Washington School of Medicine, Seattle, WA, USA;
<sup>m</sup>Department of Pathology, University of California, San Francisco, CA, USA;
<sup>n</sup>Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, USA;
<sup>o</sup>Department of Biochemistry and Biophysics, University of California, San Francisco, CA, USA.

**CONTACT** Carlo Condello carlo.condello@ucsf.edu

## ABSTRACT

We present evidence that both the Aβ and tau proteins misfold into prions leading to Alzheimer's disease (AD), which is a sporadic or genetic dementing disorder but not communicable or contagious. Since the progression of AD correlates poorly with insoluble Aβ in the central nervous system (CNS), it was difficult to distinguish between inert amyloids and Aβ prions. To measure the progression of AD, we devised exquisitely selective and sensitive bioassays to measure the abundance of isoform-specific Aβ prions in human CNS samples from patients with AD or other neurodegenerative diseases. Although brains from all human AD patients had measurable Aβ prions at death, the oldest individuals had lower Aβ prion levels. Long-lived individuals had low tau prion levels that correlated with phosphorylated tau. Surprisingly, the longevity-dependent decrease in tau prions occurred in spite of increasing amounts of total insoluble tau. When corrected for the abundance of insoluble tau, the tau prions decreased exponentially with respect to the age of death with a half-time of approximately one decade, and this correlated with the abundance of phosphorylated tau. Our findings with tau prions were not only unexpected but also counterintuitive; thus, tau phosphorylation and tau prion activity decreased exponentially with longevity
<figure><img src="image_47.png" /></figure>

difficult to diagnose. The only way to definitely diagnose prion disease is via neuropathologic examination, but reliable antemortem diagnoses can be made, especially with the advent of real time quaking induced conversion (RT-QuIC) to detect prions. Individuals with a neuropsychiatric disorder and positive RT-QuIC are considered to have probable sporadic Creutzfeldt-Jakob disease (sCJD) using current diagnostic criteria.

**Materials and Methods:** Demographic and laboratory data were examined for cerebrospinal fluid (CSF) specimens submitted to the National Prion Disease Pathology Surveillance Center (NPDPSC) from May 2015 to April 2018. In cases with multiple specimen submission, only the first specimen received was included in these analyses. Second generation RT-QuIC using truncated hamster prion protein substrate was performed on all specimens. Neuropathologic examination at autopsy was used as the gold standard comparator. Autopsy is biased in favour of cases where the diagnosis is uncertain at death, namely when false positive or false negative RT-QuIC results are suspected. Autopsy tissue received through October 2018 was included in the analyses.

**Results:** The NPDPSC received CSF specimens from 10,498 living patients with suspected prion disease, and 567 (5.4%) subsequently underwent neuropathologic examination by the NPDSPC. Overall, 1,103 (10.5%) CSF specimens submitted to the NPDPSC were RT-QuIC positive. At autopsy, 497 (87.7%) cases were positive for prion disease, including 437 (77.4%) with definitive sCJD. Overall CSF RT-QuIC diagnostic sensitivity was 90.3% (449/497) and specificity was 98.5% (69/70). Sensitivity was highest in sCJD (92.9%; 406/437) compared to genetic prion diseases (82%; 31/38). RT-QuIC was negative in all cases of sporadic fatal insomnia (sFI, *n* = 5), sCJD VV1 (*n* = 3), and fatal familial insomnia (FFI, *n* = 4). Younger subjects were more likely to have false negative RT-QuIC results. One false positive RT-QuIC test occurred in a case of multifactorial dementia (Alzheimer's disease and vascular).

**Conclusions:** We present analyses of a large cohort of CSF specimens and autopsy tissue submitted to the NPDPSC that includes high variability in demographics and prion disease subtypes. RT-QuIC demonstrated its highest sensitivity when testing CSF from older individuals and individuals with common forms of sCJD. RT-QuIC demonstrated poor sensitivity in detecting some uncommon prion disease subtypes (sFI, sCJD VV1, and FFI). The high specificity of RT-QuIC suggests that counting individuals who are positive for RT-QuIC in public health surveillance case numbers of probable/definitive prion disease, and antemortem CSF testing may foster improved prion disease surveillance.

## 68. Bioassays reveal Aβ and tau prions decline with longevity in Alzheimer's disease

Carlo Condello<sup>a,b</sup>, Atsushi Aoyagi<sup>a,c</sup>, Jan Stöhr<sup>a,b,d</sup>, Weizhou Yue<sup>a</sup>, Brianna M. Rivera<sup>a</sup>, Joanne C. Lee<sup>a</sup>, Amanda L. Woerman<sup>a,b</sup>, Glenda Halliday<sup>e</sup>, Sjoerd van Duinen<sup>f</sup>, Martin Ingelsson<sup>g</sup>, Lars Lannfelt<sup>g</sup>, Caroline Graff<sup>h,i</sup>, Thomas D. Bird<sup>j,k</sup>, C. Dirk Keene<sup>l</sup>, William W. Seeley<sup>b,m</sup>, William F. DeGrado<sup>a,n</sup> and Stanley B. Prusiner<sup>a,b,o</sup>

<sup>a</sup>Institute for Neurodegenerative Diseases, Weill Institute for Neurosciences, University of California, San Francisco, CA, USA;
<sup>b</sup>Department of Neurology, Weill Institute for Neurosciences, University of California, San Francisco, CA, USA;
<sup>c</sup>Daiichi Sankyo Co., Ltd., Tokyo, Japan;
<sup>d</sup>AC Immune SA, Lausanne, Switzerland;
<sup>e</sup>School of Medical Science, University of New South Wales, Sydney, Australia;
<sup>f</sup>Leiden University Medical Center, Leiden, The Netherlands;
<sup>g</sup>Department of Public Health and Caring Services/Geriatrics, Uppsala University, Uppsala, Sweden;
<sup>h</sup>Department of Neurobiology, Care Sciences and Society, Karolinska Institute, Huddinge, Sweden;
<sup>i</sup>Department of Geriatric Medicine, Karolinska University Hospital, Stockholm, Sweden;
<sup>j</sup>Department of Medicine, Division of Medical Genetics, University of Washington, Seattle, WA, USA;
<sup>k</sup>Department of Neurology, University of Washington, Seattle, WA, USA;
<sup>l</sup>Department of Neuropathology, University of Washington School of Medicine, Seattle, WA, USA;
<sup>m</sup>Department of Pathology, University of California, San Francisco, CA, USA;
<sup>n</sup>Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, USA;
<sup>o</sup>Department of Biochemistry and Biophysics, University of California, San Francisco, CA, USA.

**CONTACT** Carlo Condello carlo.condello@ucsf.edu

## ABSTRACT

We present evidence that both the Aβ and tau proteins misfold into prions leading to Alzheimer's disease (AD), which is a sporadic or genetic dementing disorder but not communicable or contagious. Since the progression of AD correlates poorly with insoluble Aβ in the central nervous system (CNS), it was difficult to distinguish between inert amyloids and Aβ prions. To measure the progression of AD, we devised exquisitely selective and sensitive bioassays to measure the abundance of isoform-specific Aβ prions in human CNS samples from patients with AD or other neurodegenerative diseases. Although brains from all human AD patients had measurable Aβ prions at death, the oldest individuals had lower Aβ prion levels. Long-lived individuals had low tau prion levels that correlated with phosphorylated tau. Surprisingly, the longevity-dependent decrease in tau prions occurred in spite of increasing amounts of total insoluble tau. When corrected for the abundance of insoluble tau, the tau prions decreased exponentially with respect to the age of death with a half-time of approximately one decade, and this correlated with the abundance of phosphorylated tau. Our findings with tau prions were not only unexpected but also counterintuitive; thus, tau phosphorylation and tau prion activity decreased exponentially with longevity
<figure><img src="image_44.png" /></figure>

in patients with AD ranging from ages 37 to 99 years. Our findings demonstrate an inverse correlation between longevity in AD patients and the abundance of neurotoxic tau prions. Moreover, our discovery may have profound implications for the selection of phenotypically distinct patient populations and development of diagnostics and effective therapeutics for AD.

**69. Why is there a decline in the incidence of sporadic Creutzfeldt Jakob Disease in the older adult population?**

**Investigating mixed pathologies in sporadic Creutzfeldt Jakob Disease**

Jane Lumsden, Bob Will, Colin Smith and Suvankar Pal

National CJD Research and Surveillance Unit, University of Edinburgh

**ABSTRACT**

**Background:** Sporadic Creutzfeldt Jakob Disease (sCJD) is a rapidly progressive and fatal neurodegenerative disorder and the commonest occurring form of human prion disease. Age-specific mortality rates for sCJD have increased up to ages 65–79 years over the past four decades in the United Kingdom. Of considerable interest is an apparent reduced incidence at ages of 80 and over. A similar phenomenon has been observed in other European countries. This reduced incidence could be due to an under ascertainment in the most elderly. It has also been recently hypothesized that the apparent decline in incidence of sCJD in older adults could be due to the inhibitory effects of the Alzheimer's disease associated amyloid β-protein(A β) on prion propagation. Aβ deposition is common in advanced age, even in cognitively asymptomatic individuals, and a recent study suggested that soluble aggregates of Aβ can compete with prions for binding to misfolded cellular prion protein. This study raised the possibility that presence of Alzheimer's disease pathology may be protective against CJD. Another recent neuropathological study investigating 30 cases with confirmed sCJD reported that co-morbid presence of abnormal proteins associated with other neurodegenerative disorders including Alzheimer's disease, amyloid angiopathy, Lewy body dementia, argyrophilic grain disease, and progressive supranuclear palsy was found to be significantly associated with prolonged survival. This led authors to suggest that presence of dual/multiple neurodegenerative pathologies may be contributing to this phenomenon. Furthermore, additional work has demonstrated that sCJD cases with abundant deposits of Aβ present at a higher median age of disease onset and have longer disease duration. This project proposal identifies aims and hypotheses for this ongoing project.

**Aims:**

(1) To review UK postmortem reports of patients with confirmed sCJD to establish presence of β-amyloid, neurofibrillary tangles, neuritic plaques, α synuclein, amyloid angiopathy, and record Thal phase and Braak stage.

(2) To review clinical details of these cases including clinical co-morbidities, age at onset of sCJD, presenting symptoms, disease duration, codon 129 status, MRI findings, 14-3-3, and RT-QuIC results

(3) To compare clinical features and investigation results of cases with dual/multiple co-pathologies with cases with no known co-pathology.

**Hypotheses:**

(1) Cases with neuropathological evidence of dual or multiple pathologies will present differently to cases with isolated sCJD pathology.

(2) MRI scans will show focal atrophy, or vascular burden which may confound the typical cortical and basal ganglia changes seen in sCJD.

(3) CSF 14-3-3 and RT-QuIC results will differ. Specifically, 14-3-3 will be negative in cases of dual/multiple pathologies.

(4) Individual co-pathologies will influence the clinical features and investigation results in a differential manner.

(5) Cases with significant Lewy body deposition will likely test positive for alpha-synuclein in CSF.

**70. A histidine swap in the prion protein's Copper Center Drives a neuroprotective cis-interaction**

Kevin Schillingª, Lizhi Taoᵇ, Bei Wuᶜ, Glenn Millhauserª, David Brittᵇ and David Harrisᶜ

CONTACT Kevin Schilling kschilli@ucsc.edu

**ABSTRACT**

The cellular prion protein (PrPC) is comprised of two domains – a globular C- terminal domain and an unstructured N-terminal domain. Recently, copper has been observed to drive tertiary structure in PrPC, inducing a cis-interaction between the two domains of the protein.¹–³ The location of this interaction on the C-terminus overlaps with the sites of PrPSc docking, toxic antibody docking, and pathogenic mutations. Combined with recent evidence that the N-terminus is a toxic effector regulated by the C-

<footnote>ªDepartment of Chemistry & Biochemistry, UC Santa Cruz, Santa Cruz CA, USA; ᵇDepartment of Chemistry, UC Davis, Davis, CA, USA; ᶜDepartment of Biochemistry, Boston University School of Medicine, Boston, MA, USA</footnote>
<figure><img src="image_48.png" /></figure>

in patients with AD ranging from ages 37 to 99 years. Our findings demonstrate an inverse correlation between longevity in AD patients and the abundance of neurotoxic tau prions. Moreover, our discovery may have profound implications for the selection of phenotypically distinct patient populations and development of diagnostics and effective therapeutics for AD.

**69. Why is there a decline in the incidence of sporadic Creutzfeldt Jakob Disease in the older adult population?**

**Investigating mixed pathologies in sporadic Creutzfeldt Jakob Disease**

Jane Lumsden, Bob Will, Colin Smith and Suvankar Pal

National CJD Research and Surveillance Unit, University of Edinburgh

**ABSTRACT**

**Background:** Sporadic Creutzfeldt Jakob Disease (sCJD) is a rapidly progressive and fatal neurodegenerative disorder and the commonest occurring form of human prion disease. Age-specific mortality rates for sCJD have increased up to ages 65–79 years over the past four decades in the United Kingdom. Of considerable interest is an apparent reduced incidence at ages of 80 and over. A similar phenomenon has been observed in other European countries. This reduced incidence could be due to an underascertainment in the most elderly. It has also been recently hypothesized that the apparent decline in incidence of sCJD in older adults could be due to the inhibitory effects of the Alzheimer's disease associated amyloid β-protein(A β) on prion propagation. Aβ deposition is common in advanced age, even in cognitively asymptomatic individuals, and a recent study suggested that soluble aggregates of Aβ can compete with prions for binding to misfolded cellular prion protein. This study raised the possibility that presence of Alzheimer's disease pathology may be protective against CJD. Another recent neuropathological study investigating 30 cases with confirmed sCJD reported that co-morbid presence of abnormal proteins associated with other neurodegenerative disorders including Alzheimer's disease, amyloid angiopathy, Lewy body dementia, argyrophilic grain disease, and progressive supranuclear palsy was found to be significantly associated with prolonged survival. This led authors to suggest that presence of dual/multiple neurodegenerative pathologies may be contributing to this phenomenon. Furthermore, additional work has demonstrated that sCJD cases with abundant deposits of Aβ present at a higher median age of disease onset and have longer disease duration. This project proposal identifies aims and hypotheses for this ongoing project.

**Aims:**

(1) To review UK postmortem reports of patients with confirmed sCJD to establish presence of β-amyloid, neurofibrillary tangles, neuritic plaques, α synuclein, amyloid angiopathy, and record Thal phase and Braak stage.

(2) To review clinical details of these cases including clinical co-morbidities, age at onset of sCJD, presenting symptoms, disease duration, codon 129 status, MRI findings, 14-3-3, and RT-QuIC results

(3) To compare clinical features and investigation results of cases with dual/multiple co-pathologies with cases with no known co-pathology.

**Hypotheses:**

(1) Cases with neuropathological evidence of dual or multiple pathologies will present differently to cases with isolated sCJD pathology.

(2) MRI scans will show focal atrophy, or vascular burden which may confound the typical cortical and basal ganglia changes seen in sCJD.

(3) CSF 14-3-3 and RT-QuIC results will differ. Specifically, 14-3-3 will be negative in cases of dual/multiple pathologies.

(4) Individual co-pathologies will influence the clinical features and investigation results in a differential manner.

(5) Cases with significant Lewy body deposition will likely test positive for alpha-synuclein in CSF.

**70. A histidine swap in the prion protein's Copper Center Drives a neuroprotective cis-interaction**

Kevin Schillingª, Lizhi Taoᵇ, Bei Wuᶜ, Glenn Millhauserª, David Brittᵇ and David Harrisᶜ

CONTACT Kevin Schilling kschilli@ucsc.edu

**ABSTRACT**

The cellular prion protein (PrPC) is comprised of two domains – a globular C- terminal domain and an unstructured N-terminal domain. Recently, copper has been observed to drive tertiary structure in PrPC, inducing a cis-interaction between the two domains of the protein.¹–³ The location of this interaction on the C-terminus overlaps with the sites of PrPSc docking, toxic antibody docking, and pathogenic mutations. Combined with recent evidence that the N-terminus is a toxic effector regulated by the C-

<footnote>ªDepartment of Chemistry & Biochemistry, UC Santa Cruz, Santa Cruz CA, USA; ᵇDepartment of Chemistry, UC Davis, Davis, CA, USA; ᶜDepartment of Biochemistry, Boston University School of Medicine, Boston, MA, USA</footnote>
<figure><img src="image_45.png" /></figure>

terminus,4,5 there is a significant possibility that this cis-interaction serves a protective role, and that the disruption of this interaction by PrPSc oligomers is the cause of toxicity in prion disease. We demonstrate here that two highly conserved histidines in the C-terminal domain of PrPC are essential for the protein's cis-interaction, which helps to protect against neurotoxicity carried out by its N-terminus. We show that mutation of these histidines drastically weakens the cis-interaction and biases cultured cells towards toxic events – the first C-terminal mutation to do so. Mechanistically, we propose an equatorial swap – a copper bound histidine from the N-terminus of the protein is replaced by a histidine from its C-terminus, forming a tether that holds the two domains together. We also find that extra N-terminal histidines in pathological familial mutations involving octarepeat expansions inhibit this interaction by stealing copper from the C-terminus, suggesting a mechanism for the toxicity of these mutants. We believe we have found the structural basis for PrPC's C-terminal regulation of its N-terminus, and that this mechanism is responsible for preventing neurotoxicity in the properly functioning form of the protein.

## References

[1] Thakur, et al. JBC 2011; 286:38,533–38,545.

[2] Spevacek, et al. Structure 2013; 21:236–246.

[3] Evans, et al. Structure 2016; 24:1057–10674.

[4] Sonati, et al. Nature 2013; 501:102–106.

[5] Wu, et al. eLife 2017. doi:10.7554/eLife.23,473.001

# 71. Challenging assumptions: an empirical analysis of hunter response to chronic wasting disease in Alberta

John K. Pattison-Williamsª, Lusi Xieª, Vic (W.L.) Adamowiczª, Margo Pybusb and Anne Hubbsb

CONTACT John K. Pattison-Williams johnp@ualberta.ca

## ABSTRACT

**Background:** Chronic Wasting Disease (CWD) has fundamentally impacted wildlife management in North America. An integral partner in CWD management strategies is the hunting community, whose membership contributes in surveillance, harvest, and policy development. A conventional assumption has been that hunters will avoid or reduce hunting in areas where CWD is identified as high prevalence due to potential human health risks [1], requiring policy incentives to promote

continued or increased hunting in these areas. However, this assumption has recently been challenged [2].

**Methods:** Using mule deer hunter draw information and CWD surveillance data from the Alberta Ministry of Environment and Parks (AEP), we empirically explore the response of mule deer hunters to CWD using license application trends in wildlife management units (WMUs) where CWD has been positively detected. Using a fixed effects (FE) approach, we model the relationship between total resident draw applications and covariates of CWD head counts, quotas, draw success rates between 2005 and 2016 in 39 WMUs.

**Results:** Our model indicates that mule deer hunters in Alberta are continuing to hunt in WMUs with high CWD prevalence and no significant relationship exists between CWD count and draw applications. These results suggest that (i) hunter decisions regarding license applications are not significantly affected by CWD presence or prevalence, or (ii) hunters perceive CWD in a WMU as a factor that does not detract from, and may be attractive for hunting opportunities.

**Conclusions:** Our results have important policy implications for CWD management. First, it challenges the assumption that hunters are avoiding CWD zones. Second, it suggests that alternative hunter-based strategies – such as expanded hunting seasons, increased tags or replacement tags – may be more effective and less costly than previously thought to manage the disease. One caveat is that our analysis is based on analyses of aggregate demand for hunting licenses at the WMU level, rather than individual hunter demands over time.

**KEYWORDS:** Chronic wasting disease; recreational hunting; cervids; alberta; mule deer; wildlife management

## References

[1] Cooney, et al. Hum Dimens Wildl 2010; 15: 194–207.

[2] Haus, et al. Wildl Soc Bull 2017; 41:294–300.

# 72. miR-146a regulates cellular and disease associated prion protein isoforms during prion propagation

Wenting Zhaoa,b, Cathryn Ugaldeª, Steven Collinsc, Vicki Lawsond, Lesley Chengª, Shayne Bellinghamb and Andrew Hillª

<footnote>ªDepartment of Resource Economics and Environmental Sociology, University of Alberta, Edmonton, Alberta, Canada; ᵇAlberta Environment and Parks, Edmonton, Alberta, Canada</footnote>

<footnote>ªDepartment of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, VIC, Australia; ᵇDepartment of Biochemistry and Molecular Biology, The University of Melbourne, VIC, Australia; cFlorey Institute of Neuroscience and Mental Health, The University of Melbourne, VIC, Australia; dDepartment of Microbiology</footnote>
<figure><img src="image_49.png" /></figure>

terminus,4,5 there is a significant possibility that this cis-interaction serves a protective role, and that the disruption of this interaction by PrPSc oligomers is the cause of toxicity in prion disease. We demonstrate here that two highly conserved histidines in the C-terminal domain of PrPC are essential for the protein's cis-interaction, which helps to protect against neurotoxicity carried out by its N-terminus. We show that mutation of these histidines drastically weakens the cis-interaction and biases cultured cells towards toxic events – the first C-terminal mutation to do so. Mechanistically, we propose an equatorial swap – a copper bound histidine from the N-terminus of the protein is replaced by a histidine from its C-terminus, forming a tether that holds the two domains together. We also find that extra N-terminal histidines in pathological familial mutations involving octarepeat expansions inhibit this interaction by stealing copper from the C-terminus, suggesting a mechanism for the toxicity of these mutants. We believe we have found the structural basis for PrPC's C-terminal regulation of its N-terminus, and that this mechanism is responsible for preventing neurotoxicity in the properly functioning form of the protein.

## References

[1] Thakur, et al. JBC 2011; 286:38,533–38,545.

[2] Spevacek, et al. Structure 2013; 21:236–246.

[3] Evans, et al. Structure 2016; 24:1057–10674.

[4] Sonati, et al. Nature 2013; 501:102–106.

[5] Wu, et al. eLife 2017. doi:10.7554/eLife.23,473.001

# 71. Challenging assumptions: an empirical analysis of hunter response to chronic wasting disease in Alberta

John K. Pattison-Williamsª, Lusi Xieª, Vic (W.L.) Adamowiczª, Margo Pybusb and Anne Hubbsb

CONTACT John K. Pattison-Williams johnp@ualberta.ca

## ABSTRACT

**Background:** Chronic Wasting Disease (CWD) has fundamentally impacted wildlife management in North America. An integral partner in CWD management strategies is the hunting community, whose membership contributes in surveillance, harvest, and policy development. A conventional assumption has been that hunters will avoid or reduce hunting in areas where CWD is identified as high prevalence due to potential human health risks [1], requiring policy incentives to promote

continued or increased hunting in these areas. However, this assumption has recently been challenged [2].

**Methods:** Using mule deer hunter draw information and CWD surveillance data from the Alberta Ministry of Environment and Parks (AEP), we empirically explore the response of mule deer hunters to CWD using license application trends in wildlife management units (WMUs) where CWD has been positively detected. Using a fixed effects (FE) approach, we model the relationship between total resident draw applications and covariates of CWD head counts, quotas, draw success rates between 2005 and 2016 in 39 WMUs.

**Results:** Our model indicates that mule deer hunters in Alberta are continuing to hunt in WMUs with high CWD prevalence and no significant relationship exists between CWD count and draw applications. These results suggest that (i) hunter decisions regarding license applications are not significantly affected by CWD presence or prevalence, or (ii) hunters perceive CWD in a WMU as a factor that does not detract from, and may be attractive for hunting opportunities.

**Conclusions:** Our results have important policy implications for CWD management. First, it challenges the assumption that hunters are avoiding CWD zones. Second, it suggests that alternative hunter-based strategies – such as expanded hunting seasons, increased tags or replacement tags – may be more effective and less costly than previously thought to manage the disease. One caveat is that our analysis is based on analyses of aggregate demand for hunting licenses at the WMU level, rather than individual hunter demands over time.

**KEYWORDS:** Chronic wasting disease; recreational hunting; cervids; alberta; mule deer; wildlife management

## References

[1] Cooney, et al. Hum Dimens Wildl 2010; 15: 194–207.

[2] Haus, et al. Wildl Soc Bull 2017; 41:294–300.

# 72. miR-146a regulates cellular and disease associated prion protein isoforms during prion propagation

Wenting Zhaoa,b, Cathryn Ugaldeª, Steven Collinsc, Vicki Lawsond, Lesley Chengª, Shayne Bellinghamb and Andrew Hillª

<footnote>ªDepartment of Resource Economics and Environmental Sociology, University of Alberta, Edmonton, Alberta, Canada; ᵇAlberta Environment and Parks, Edmonton, Alberta, Canada</footnote>

<footnote>ªDepartment of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, VIC, Australia; ᵇDepartment of Biochemistry and Molecular Biology, The University of Melbourne, VIC, Australia; cFlorey Institute of Neuroscience and Mental Health, The University of Melbourne, VIC, Australia; dDepartment of Microbiology</footnote>
<figure><img src="image_46.png" /></figure>

and Immunology, The Peter Doherty Institute for Infection and
Immunity, The University of Melbourne, VIC, Australia.

CONTACT Wenting Zhao wenting.zhao@latrobe.edu.au

ABSTRACT

Prion diseases are transmissible neurodegenerative
disorders characterized by the structural transforma-
tion of the cellular prion protein (PrP<sup>C</sup>) to the prion
disease associated isoform (PrP<sup>Sc</sup>). MicroRNAs
(miRNAs) are a class of small non-coding RNAs
that regulate gene or protein expression by targeting
mRNAs and triggering either translational repression
or mRNA degradation. Distinct miRNA signatures
have been detected in prion disease models and
patients, serving as potential disease diagnostic bio-
markers. In this study, increased expression levels of
miR-146a, an anti-inflammatory miRNA known to be
dysregulated in prion diseases, were detected in four
prion disease models: in prion strain M1000 infected
neuronal N2a cells; at day 42 and day 70 of M1000
infected organotypic brain slices; at week 17 and
terminal stages of M1000 infected mouse brains;
and in the post-mortem brain tissue samples from
sporadic Creutzfeldt-Jakob disease (sCJD) patients.
CRISPR/Cas9 mediated miRNA editing and prion
infection were further used to examine the effects of
miR-146a gain/loss-of-function on prion proteins in
cell culture and organotypic brain slice culture mod-
els. miR-146a was shown to increase PrP<sup>C</sup> levels
when overexpressed or downregulated in N2a cells.
The prion protein encoding gene (Prnp) was shown
to be targeted by miR-146a in the 3' untranslated
region (3' UTR). miR-146a overexpression decreased
the amount of PrP<sup>Sc</sup> in M1000 infected N2a cells,
while M1000 infected organotypic brain slices from
miR-146a knockout mice exhibited increased PrP<sup>Sc</sup>
levels compared to the wildtype controls. Overall,
the protective role of miR-146a was revealed in
prion diseases by regulating cellular and disease asso-
ciated prion proteins, suggesting its therapeutic
potential.

73. Transgenic overexpression of the PrP N1
fragment does not protect against prion disease or
Aβ toxicity due to impaired ER translocation

Behnam Mohammadiª, Luise Linsenmeierª, Mohsin
Shafiqª, Berta Puigª, Giovanna Galliciottiª, Jörg
Tatzeltᵇ, Markus Glatzelª* and Hermann C. Altmeppenª*

CONTACT Behnam Mohammadi b.mohammadi@uke.de

*These authors contributed equally to this work.

ABSTRACT

The highly conserved and constitutively active endogenous α-cleavage of the prion protein has so far mostly been linked with protective effects. As a soluble factor, the released unstructured N1 fragment acts beneficial in several ways as it, for instance, reduces hypoxia-induced neuronal damage and may be involved in myelin maintenance and other aspects of intercellular communication. With regard to neurodegenerative diseases, numerous studies have shown that N1 is able to block toxic oligomers, such as Aβ in Alzheimer's disease, and interferes with their synaptic impairment and neurotoxicity.

For prion diseases, however, insight into a poten-
tially protective role of N1 by similarly neutralizing
PrP<sup>Sc</sup> oligomers and interfering with prion conversion
is lacking to date. Since the responsible protease for the
α-cleavage has not been identified unequivocally, phar-
macological targeting of this entity is not possible to
date. We therefore decided to directly address this issue
*in vivo* by generating transgenic mice (tgN1) overex-
pressing N1 on a wild-type background and challenging
them with RML prions. Despite moderate differences in
PrP<sup>Sc</sup> formation and p38 activation, incubation time,
and disease duration was similar between tgN1 mice
and wild-type littermate controls. Biochemical and
morphological assessment of brain samples, primary
neurons, and cell culture models then revealed, that
our direct 'protective' strategy failed due to cytosolic
accumulation and lack of secretion of the transgenic
N1. Nevertheless, our study provides the first *in vivo*
proof of the recently described impaired translocation
of intrinsically disordered peptides into the endoplas-
mic reticulum. Moreover, it demonstrates effects of
cytosolic accumulation of N1 with uncleaved signal
peptide, addresses proteasomal degradation, questions
a general relevance of cytosolic prions in prion diseases,
and highlights important aspects to be considered when
investigating the α-cleavage of PrP<sup>C</sup> or devising N1-
based therapeutic approaches.

74. Characterization of gene expression profiles in
brains of mice infected with typical and atypical
BSEs

Takeshi Yamasakiª, Akio Suzukiª, Rie Hasebeᵇ, Yuichi
Matsuuraᶜ, Kohtaro Miyazawaᶜ, Yoshifumi Iwamaruᶜ
and Motohiro Horiuchiª

<footnote>ªInstitute of Neuropathology, University Medical Center Hamburg-Eppendorf (UKE), Hamburg, Germany; ᵇBiochemistry of Neurodegenerative Diseases, Institute of Biochemistry and Pathobiochemistry, Ruhr University Bochum, Bochum, Germany</footnote>

<footnote>ªLaboratory of Veterinary Hygiene, Faculty of Veterinary Medicine, Hokkaido University, Japan; ᵇInstitute for Genetic Medicine,</footnote>
<figure><img src="image_50.png" /></figure>

and Immunology, The Peter Doherty Institute for Infection and
Immunity, The University of Melbourne, VIC, Australia.

CONTACT Wenting Zhao wenting.zhao@latrobe.edu.au

ABSTRACT

Prion diseases are transmissible neurodegenerative
disorders characterized by the structural transforma-
tion of the cellular prion protein (PrP<sup>C</sup>) to the prion
disease associated isoform (PrP<sup>Sc</sup>). MicroRNAs
(miRNAs) are a class of small non-coding RNAs
that regulate gene or protein expression by targeting
mRNAs and triggering either translational repression
or mRNA degradation. Distinct miRNA signatures
have been detected in prion disease models and
patients, serving as potential disease diagnostic bio-
markers. In this study, increased expression levels of
miR-146a, an anti-inflammatory miRNA known to be
dysregulated in prion diseases, were detected in four
prion disease models: in prion strain M1000 infected
neuronal N2a cells; at day 42 and day 70 of M1000
infected organotypic brain slices; at week 17 and
terminal stages of M1000 infected mouse brains;
and in the post-mortem brain tissue samples from
sporadic Creutzfeldt-Jakob disease (sCJD) patients.
CRISPR/Cas9 mediated miRNA editing and prion
infection were further used to examine the effects of
miR-146a gain/loss-of-function on prion proteins in
cell culture and organotypic brain slice culture mod-
els. miR-146a was shown to increase PrP<sup>C</sup> levels
when overexpressed or downregulated in N2a cells.
The prion protein encoding gene (Prnp) was shown
to be targeted by miR-146a in the 3' untranslated
region (3' UTR). miR-146a overexpression decreased
the amount of PrP<sup>Sc</sup> in M1000 infected N2a cells,
while M1000 infected organotypic brain slices from
miR-146a knockout mice exhibited increased PrP<sup>Sc</sup>
levels compared to the wildtype controls. Overall,
the protective role of miR-146a was revealed in
prion diseases by regulating cellular and disease asso-
ciated prion proteins, suggesting its therapeutic
potential.

73. Transgenic overexpression of the PrP N1
fragment does not protect against prion disease or
Aβ toxicity due to impaired ER translocation

Behnam Mohammadiª, Luise Linsenmeierª, Mohsin Shafiqª, Berta Puigª, Giovanna Galliciottiª, Jörg Tatzeltᵇ, Markus Glatzelª* and Hermann C. Altmeppenª*

CONTACT Behnam Mohammadi b.mohammadi@uke.de

*These authors contributed equally to this work.

ABSTRACT

The highly conserved and constitutively active endogenous α-cleavage of the prion protein has so far mostly been linked with protective effects. As a soluble factor, the released unstructured N1 fragment acts beneficial in several ways as it, for instance, reduces hypoxia-induced neuronal damage and may be involved in myelin maintenance and other aspects of intercellular communication. With regard to neurodegenerative diseases, numerous studies have shown that N1 is able to block toxic oligomers, such as Aβ in Alzheimer's disease, and interferes with their synaptic impairment and neurotoxicity.

For prion diseases, however, insight into a poten-
tially protective role of N1 by similarly neutralizing
PrP<sup>Sc</sup> oligomers and interfering with prion conversion
is lacking to date. Since the responsible protease for the
α-cleavage has not been identified unequivocally, phar-
macological targeting of this entity is not possible to
date. We therefore decided to directly address this issue
*in vivo* by generating transgenic mice (tgN1) overex-
pressing N1 on a wild-type background and challenging
them with RML prions. Despite moderate differences in
PrP<sup>Sc</sup> formation and p38 activation, incubation time,
and disease duration was similar between tgN1 mice
and wild-type littermate controls. Biochemical and
morphological assessment of brain samples, primary
neurons, and cell culture models then revealed, that
our direct 'protective' strategy failed due to cytosolic
accumulation and lack of secretion of the transgenic
N1. Nevertheless, our study provides the first *in vivo*
proof of the recently described impaired translocation
of intrinsically disordered peptides into the endoplas-
mic reticulum. Moreover, it demonstrates effects of
cytosolic accumulation of N1 with uncleaved signal
peptide, addresses proteasomal degradation, questions
a general relevance of cytosolic prions in prion diseases,
and highlights important aspects to be considered when
investigating the α-cleavage of PrP<sup>C</sup> or devising N1-
based therapeutic approaches.

74. Characterization of gene expression profiles in
brains of mice infected with typical and atypical
BSEs

Takeshi Yamasakiª, Akio Suzukiª, Rie Hasebeᵇ, Yuichi Matsuuraᶜ, Kohtaro Miyazawaᶜ, Yoshifumi Iwamaruᶜ and Motohiro Horiuchiª

<footnote>ªInstitute of Neuropathology, University Medical Center Hamburg-Eppendorf (UKE), Hamburg, Germany; ᵇBiochemistry of Neurodegenerative Diseases, Institute of Biochemistry and Pathobiochemistry, Ruhr University Bochum, Bochum, Germany</footnote>

<footnote>ªLaboratory of Veterinary Hygiene, Faculty of Veterinary Medicine, Hokkaido University, Japan; ᵇInstitute for Genetic Medicine,</footnote>
<figure><img src="image_47.png" /></figure>

Hokkaido University, Japan; ´National Institute of Animal Health, National Agriculture and Food Research Organization (NARO), Japan

CONTACT Takeshi Yamasaki Yamasaki@vetmed.hokudai.ac.jp

ABSTRACT

Transgenic mice overexpressing bovine PrP (TgBoPrP) infected with typical BSE (C-BSE), atypical L-type BSE, and H-type BSE (L-BSE and H-BSE) show different clinical and neuropathological characteristics. To understand host responses that contribute to the differences in the phenotypes, we analysed gene expression profiles in brains of TgBoPrP mice infected with C-BSE, L-BSE, and H-BSE. We performed a transcriptome analysis by RNA-sequencing on brainstems of the mice at pre-clinical, clinical, and terminal stages of infection and those of age-matched uninfected mice. First, we performed hierarchical clustering of samples based on gene expression data using 16,894 genes that code proteins. The samples from mice infected with L-BSE at 198 days post intracerebral inoculation (dpi), C-BSE at 239 and 253 dpi, and H-BSE at 291, 319 and 362 dpi were clearly separated from cluster containing un-infected samples, suggesting that host responses accompanied with marked transcriptomic changes initiated by 198, 239, and 291 dpi in L-BSE, C-BSE and H-BSE infected mice, respectively. Next, we identified 3,219 differentially expressed genes (DEGs) that showed over 2-fold change in expression level between infected and un-infected mice. By applying K-means clustering to the DEGs, we found four characteristic gene clusters that contain genes of which expression was upregulated by infection. Three clusters contained 338, 517 and 294 upregulated genes unique to C-BSE, L-BSE, and H-BSE, respectively, and the other cluster contained 251 upregulated genes common to all BSEs. Gene ontology (GO) analysis based on Metascape database revealed that genes related to immune responses were highly enriched in the upregulated genes common to all BSEs. Although enrichment scores were not so high, the upregulated genes unique to each BSE showed a significant enrichment of GO terms related to regulation of immune system. These data might suggest the presence of immune responses both common and unique to C-BSE, L-BSE, and H-BSE infection.

75. Mortality surveillance of individuals potentially exposed to chronic wasting disease

Ryan A. Maddox<sup>a</sup>, Rachel F. Klos<sup>b</sup>, Suzanne N. Gibbons-Burgener<sup>b</sup>, Bobbi L. Bryant<sup>a</sup>, Joseph Y. Abrams<sup>a</sup>, Brian S. Appleby<sup>c</sup>, Lawrence B. Schonberger<sup>a</sup> and Ermias D. Belay<sup>a</sup>

<sup>a</sup>National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention (CDC), Atlanta, GA, USA; <sup>b</sup>Wisconsin Department of Health Services, Division of Public Health, Madison, WI, USA; <sup>c</sup>National Prion Disease Pathology Surveillance Center (NPDPSC), Case Western Reserve University, Cleveland, OH, USA

CONTACT Ryan A. Maddox rmaddox@cdc.gov

ABSTRACT

**Introduction:** Chronic wasting disease (CWD) is a prion disease of cervids. It is unknown whether CWD prions can infect people; if so, transmission would most likely occur through consumption of meat from infected animals. Since 2003, Wisconsin Department of Health Services, Division of Public Health (WDHS) personnel have maintained a database consisting of information collected from hunters who reported eating, or an intention to eat, venison from cervids positive for CWD. This data source makes it possible to evaluate causes of mortality in individuals potentially exposed to CWD.

**Methods:** The WDHS database contains the name, date of birth, when available, year of CWD-positive deer harvest, and city and state of residence for each potentially exposed individual. The database also includes information on how the deer was processed (self-processed or by a commercial operator) and when applicable, names of others with whom the venison was shared. Duplicate entries (i.e. those who consumed venison from CWD-positive deer in multiple hunt years) are determined by first name, last name, and date of birth. All names in the database are cross-checked with the National Prion Disease Pathology Surveillance Center (NPDPSC) neuropathology database. Vital status of individuals with date of birth available will be tracked through the identification of possible matches in the National Death Index (NDI) and the evaluation of corresponding cause of death codes.

**Results:** The database consists of 1561 records for hunt years 2003–2017. Of these, 613 records had accompanying date of birth; 14 entries were removed as duplicates, 1 of whom had consumed venison from a CWD-positive deer during three different hunt years, leaving 599 unique individuals for pending submission to NDI. Of these individuals, 265 of 399 (66%) who ate venison from a CWD-positive deer and provided processing information reported self-processing. No matches were found among persons in the database cross-checked with NPDPSC data.

**Conclusion:** Because of the robust data link between person and CWD-positive animal, reviewing the cause of mortality in potentially exposed persons is possible; those individuals who self-processed and consumed the meat are
<figure><img src="image_51.png" /></figure>

Hokkaido University, Japan; ´National Institute of Animal Health, National Agriculture and Food Research Organization (NARO), Japan

CONTACT Takeshi Yamasaki Yamasaki@vetmed.hokudai.ac.jp

ABSTRACT

Transgenic mice overexpressing bovine PrP (TgBoPrP) infected with typical BSE (C-BSE), atypical L-type BSE, and H-type BSE (L-BSE and H-BSE) show different clinical and neuropathological characteristics. To understand host responses that contribute to the differences in the phenotypes, we analysed gene expression profiles in brains of TgBoPrP mice infected with C-BSE, L-BSE, and H-BSE. We performed a transcriptome analysis by RNA-sequencing on brainstems of the mice at pre-clinical, clinical, and terminal stages of infection and those of age-matched uninfected mice. First, we performed hierarchical clustering of samples based on gene expression data using 16,894 genes that code proteins. The samples from mice infected with L-BSE at 198 days post intracerebral inoculation (dpi), C-BSE at 239 and 253 dpi, and H-BSE at 291, 319 and 362 dpi were clearly separated from cluster containing un-infected samples, suggesting that host responses accompanied with marked transcriptomic changes initiated by 198, 239, and 291 dpi in L-BSE, C-BSE and H-BSE infected mice, respectively. Next, we identified 3,219 differentially expressed genes (DEGs) that showed over 2-fold change in expression level between infected and un-infected mice. By applying K-means clustering to the DEGs, we found four characteristic gene clusters that contain genes of which expression was upregulated by infection. Three clusters contained 338, 517 and 294 upregulated genes unique to C-BSE, L-BSE, and H-BSE, respectively, and the other cluster contained 251 upregulated genes common to all BSEs. Gene ontology (GO) analysis based on Metascape database revealed that genes related to immune responses were highly enriched in the upregulated genes common to all BSEs. Although enrichment scores were not so high, the upregulated genes unique to each BSE showed a significant enrichment of GO terms related to regulation of immune system. These data might suggest the presence of immune responses both common and unique to C-BSE, L-BSE, and H-BSE infection.

75. Mortality surveillance of individuals potentially exposed to chronic wasting disease

Ryan A. Maddox<sup>a</sup>, Rachel F. Klos<sup>b</sup>, Suzanne N. Gibbons-Burgener<sup>b</sup>, Bobbi L. Bryant<sup>a</sup>, Joseph Y. Abrams<sup>a</sup>, Brian S. Appleby<sup>c</sup>, Lawrence B. Schonberger<sup>a</sup> and Ermias D. Belay<sup>a</sup>

<sup>a</sup>National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention (CDC), Atlanta, GA, USA; <sup>b</sup>Wisconsin Department of Health Services, Division of Public Health, Madison, WI, USA; <sup>c</sup>National Prion Disease Pathology Surveillance Center (NPDPSC), Case Western Reserve University, Cleveland, OH, USA

CONTACT Ryan A. Maddox rmaddox@cdc.gov

ABSTRACT

**Introduction:** Chronic wasting disease (CWD) is a prion disease of cervids. It is unknown whether CWD prions can infect people; if so, transmission would most likely occur through consumption of meat from infected animals. Since 2003, Wisconsin Department of Health Services, Division of Public Health (WDHS) personnel have maintained a database consisting of information collected from hunters who reported eating, or an intention to eat, venison from cervids positive for CWD. This data source makes it possible to evaluate causes of mortality in individuals potentially exposed to CWD.

**Methods:** The WDHS database contains the name, date of birth, when available, year of CWD-positive deer harvest, and city and state of residence for each potentially exposed individual. The database also includes information on how the deer was processed (self-processed or by a commercial operator) and when applicable, names of others with whom the venison was shared. Duplicate entries (i.e. those who consumed venison from CWD-positive deer in multiple hunt years) are determined by first name, last name, and date of birth. All names in the database are cross-checked with the National Prion Disease Pathology Surveillance Center (NPDPSC) neuropathology database. Vital status of individuals with date of birth available will be tracked through the identification of possible matches in the National Death Index (NDI) and the evaluation of corresponding cause of death codes.

**Results:** The database consists of 1561 records for hunt years 2003–2017. Of these, 613 records had accompanying date of birth; 14 entries were removed as duplicates, 1 of whom had consumed venison from a CWD-positive deer during three different hunt years, leaving 599 unique individuals for pending submission to NDI. Of these individuals, 265 of 399 (66%) who ate venison from a CWD-positive deer and provided processing information reported self-processing. No matches were found among persons in the database cross-checked with NPDPSC data.

**Conclusion:** Because of the robust data link between person and CWD-positive animal, reviewing the cause of mortality in potentially exposed persons is possible; those individuals who self-processed and consumed the meat are