<figure><img src="image_124.png" /></figure>

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

## Reference

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

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

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

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

CONTACT Alsu Kuznetsova alsu@ualberta.ca

## ABSTRACT

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

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

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

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

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

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

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

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

CONTACT Danielle Gushue dgushue@ualberta.ca

## ABSTRACT

Chronic wasting disease (CWD) is a contagious prion disease of free-ranging and captive cervids from North America, Scandinavia, and Korea. The transmission cycle of CWD among cervids relies on the lymphotropic nature of CWD prions and the environmental persistence of prion infectivity shed by affected animals. Multiple prion strains circulate in CWD enzootic areas and their diversity is modulated by disease transmission between cervids expressing different PrPC molecules. Prion strains neuroinvade with different efficiency and this process is dependent on the route of exposure, the infectious dose and the structural compatibility between prion conformation and host PrPC. To evaluate if the route of exposure affects strain adaptation, we compared the neuroinvasion properties of pure Wisc-1 strain and a mixture of this one with the H95<sup>+</sup> strain. We previously demonstrated these strains have distinct biological properties, including conformational differences, brain targeting areas, and transmission host range. Two transgenic mouse lines expressing PrPC allelotypes, Q95G96 or Q95S96, were exposed either orally or intraperitoneally (IP) and the phenotypic properties produced by each strain-route-host combination were compared. Consistent with previous observations in host expressing wt PrPC, the Wisc-1 strain had higher disease penetrance and shorter incubation periods compared to the H95<sup>+</sup>. The intraperitoneal route was more efficient than the oral route, which lacked complete attack rates. The incubation period of Wisc-1 doubled compared to the intracranial (IC) route. Interestingly, the differences in incubation between the host exposed to Wisc-1 or the H95<sup>+</sup> mixture were smaller than previously reported by IC route. The Wisc-1 host-specific titre in the H95<sup>+</sup> mixture is at least two-fold lower than pure Wisc-1, suggesting the route of exposure influenced strain selection from the mixture. This also indicates Wisc-1 is
<figure><img src="image_121.png" /></figure>

highly adapted for neuroinvasion of deer expressing wt-
PrP<sup>C</sup>. With the potential for new prion strains to emerge
and the presence of multiple strains in a single host, it is
important to characterize their transmission properties,
including their capacity to neuroinvade and their tissue
tropism.

**210. Neurofilament light chain (NfL) as a possible biomarker for drug efficacy in mouse models of neurodegenerative diseases**

Masakazu Hirouchi<sup>a,b*</sup>, Atsushi Aoyagi<sup>a,b</sup>, Annalise Bond<sup>a</sup>, Kurt Giles<sup>a,c</sup>, Carlo Condello<sup>a,c</sup> and Stanley B. Prusiner<sup>a,c</sup>

CONTACT Masakazu Hirouchi hirouchi.masakazu.m7@daiichisan-kyo.co.jp

ABSTRACT

Neurofilament light chain (NfL) is known as a promising
fluid biomarker of neurodegenerative disease progres-
sion. NfL changes in plasma have been found in humans
with neurodegenerative diseases (NDs) and transgenic
(Tg) mouse models. Here, we report an elevation of
plasma NfL in Tg mice modelling a synucleinopathy
caused by multiple system atrophy (MSA). Brain extracts
from deceased MSA patients caused disease in Tg
(SNCA\*A53T) mice expressing mutant α-synuclein
(A53T). NfL levels in plasma correlated with α-synuclein
prions and phosphorylated α-synuclein in the brain. By
measuring NfL in plasma, we were able to predict the life
span of Tg(SNCA\*A53T) mice inoculated with MSA
brain homogenates. Our findings suggest that plasma
NfL levels can be used to predict disease progression in
Tg mouse models of NDs. Preclinical methods for mea-
suring neurodegeneration in Tg mouse models are valu-
able systems for assessing efficacy in therapeutic studies.
To investigate this idea further, we employed a PrP
disease model in which RML prions are used to infect
FVB mice, which were treated with the aminothiazole
IND24, a previously published anti-prion compound.
Plasma NfL levels increased in inoculated FVB mice
without treatment, but NfL was suppressed in mice
treated with IND24, which was consistent with their
extended survival. We are currently measuring PrP<sup>Sc</sup> in
brain and NfL in plasma levels in scrapie prion-infected
FVB mice to understand the relationship between drug
target and biomarker in our study.

**211. Prion infection dysregulates retromer mediated trafficking**

Govinda Sharma, Li Lu and Sabine Gilch

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

CONTACT Govinda Sharma govinda.sharma@ucalgary.ca

ABSTRACT

**Background:** Vesicle trafficking is impaired in most of the neurodegenerative diseases caused by protein misfolding. We have previously reported that membrane bound Rab7 is reduced in prion infected cells. Rab7 regulates the trafficking from early endosome to late endosomes. Similarly, retromer complex mediates the trafficking between endosomes and the Golgi apparatus. It has been reported that the VPS35, a component of retromer complex, is downregulated in the brains of Creutzfeldt Jacob disease (CJD) patients. Here we studied how prion infection affects the retromer based trafficking in cultured cells and analysed the levels of VPS35, in prion infected mice as well as cultured cells.

**Methods:** Non-infected or prion-infected (22L-CAD5) cells were allowed to bind fluorescently labeled Cholera toxin subunit B (CTB) for 30 min at 4°C. Then the transport of CTB was chased at 37°C at different time points followed by immunostaining with Rab5 or Rab6 to visualize early endosomes or the Golgi complex, respectively. Confocal microscopy (Zeiss LSM700) and the software Zen were used to analyse the colocalization of CTB with Rab5 or Rab6. Similarly, the intracellular localization of VPS35 in 22L- or non-infected Neuro2A (N2A) was studied using immunofluorescent staining and confocal microscopy. Prion infection was confirmed by assessing the presence of proteinase K (PK) resistant prion protein (PrPSc) in cell lysates. Western blot analysis was used to evaluate the levels of VPS35 in non-infected or 22L-N2A cells, as well as in brains of 22L prion- or mock (PBS)-inoculated mice 125 days post intracerebral infection (dpi).

**Results:** We observed that GM1, a sphingolipid present in the lipid raft, is upregulated in prion-infected cells. CTB binds to GM1 and follows the endocytic route to the Golgi via retromer-mediated transport. We found that CTB transport from Rab5 positive early endosomes to the Golgi is delayed in prion-infected CAD5 as well as N2A cells. Also, the intracellular localization of the retromer component VPS35 is different in prion-infected N2A cells when compared to non-infected cells. It has been reported that VPS35 levels are downregulated in CJD patient's brain. We found a similar decrease in VPS35 levels in 22L-CAD5 cells as well as in brains of mice

<footnote><sup>a</sup>Institute for Neurodegenerative Diseases, Weill Institute for Neurosciences, University of California, San Francisco, CA, USA;</footnote>

<footnote><sup>a</sup>Daiichi Sankyo Co., Ltd, Tokyo, Japan; <sup>c</sup>Department of Neurology, University of California, San Francisco, CA, USA</footnote>
<figure><img src="image_125.png" /></figure>

highly adapted for neuroinvasion of deer expressing wt-
PrP<sup>C</sup>. With the potential for new prion strains to emerge
and the presence of multiple strains in a single host, it is
important to characterize their transmission properties,
including their capacity to neuroinvade and their tissue
tropism.

**210. Neurofilament light chain (NfL) as a possible biomarker for drug efficacy in mouse models of neurodegenerative diseases**

Masakazu Hirouchi<sup>a,b*</sup>, Atsushi Aoyagi<sup>a,b</sup>, Annalise Bond<sup>a</sup>, Kurt Giles<sup>a,c</sup>, Carlo Condello<sup>a,c</sup> and Stanley B. Prusiner<sup>a,c</sup>

CONTACT Masakazu Hirouchi hirouchi.masakazu.m7@daiichisan-kyo.co.jp

ABSTRACT

Neurofilament light chain (NfL) is known as a promising
fluid biomarker of neurodegenerative disease progres-
sion. NfL changes in plasma have been found in humans
with neurodegenerative diseases (NDs) and transgenic
(Tg) mouse models. Here, we report an elevation of
plasma NfL in Tg mice modelling a synucleinopathy
caused by multiple system atrophy (MSA). Brain extracts
from deceased MSA patients caused disease in Tg
(SNCA\*A53T) mice expressing mutant α-synuclein
(A53T). NfL levels in plasma correlated with α-synuclein
prions and phosphorylated α-synuclein in the brain. By
measuring NfL in plasma, we were able to predict the life
span of Tg(SNCA\*A53T) mice inoculated with MSA
brain homogenates. Our findings suggest that plasma
NfL levels can be used to predict disease progression in
Tg mouse models of NDs. Preclinical methods for mea-
suring neurodegeneration in Tg mouse models are valu-
able systems for assessing efficacy in therapeutic studies.
To investigate this idea further, we employed a PrP
disease model in which RML prions are used to infect
FVB mice, which were treated with the aminothiazole
IND24, a previously published anti-prion compound.
Plasma NfL levels increased in inoculated FVB mice
without treatment, but NfL was suppressed in mice
treated with IND24, which was consistent with their
extended survival. We are currently measuring PrP<sup>Sc</sup> in
brain and NfL in plasma levels in scrapie prion-infected
FVB mice to understand the relationship between drug
target and biomarker in our study.

**211. Prion infection dysregulates retromer mediated trafficking**

Govinda Sharma, Li Lu and Sabine Gilch

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

CONTACT Govinda Sharma govinda.sharma@ucalgary.ca

ABSTRACT

**Background:** Vesicle trafficking is impaired in most of the neurodegenerative diseases caused by protein misfolding. We have previously reported that membrane bound Rab7 is reduced in prion infected cells. Rab7 regulates the trafficking from early endosome to late endosomes. Similarly, retromer complex mediates the trafficking between endosomes and the Golgi apparatus. It has been reported that the VPS35, a component of retromer complex, is downregulated in the brains of Creutzfeldt Jacob disease (CJD) patients. Here we studied how prion infection affects the retromer based trafficking in cultured cells and analysed the levels of VPS35, in prion infected mice as well as cultured cells.

**Methods:** Non-infected or prion-infected (22L-CAD5) cells were allowed to bind fluorescently labeled Cholera toxin subunit B (CTB) for 30 min at 4°C. Then the transport of CTB was chased at 37°C at different time points followed by immunostaining with Rab5 or Rab6 to visualize early endosomes or the Golgi complex, respectively. Confocal microscopy (Zeiss LSM700) and the software Zen were used to analyse the colocalization of CTB with Rab5 or Rab6. Similarly, the intracellular localization of VPS35 in 22L- or non-infected Neuro2A (N2A) was studied using immunofluorescent staining and confocal microscopy. Prion infection was confirmed by assessing the presence of proteinase K (PK) resistant prion protein (PrPSc) in cell lysates. Western blot analysis was used to evaluate the levels of VPS35 in non-infected or 22L-N2A cells, as well as in brains of 22L prion- or mock (PBS)-inoculated mice 125 days post intracerebral infection (dpi).

**Results:** We observed that GM1, a sphingolipid present in the lipid raft, is upregulated in prion-infected cells. CTB binds to GM1 and follows the endocytic route to the Golgi via retromer-mediated transport. We found that CTB transport from Rab5 positive early endosomes to the Golgi is delayed in prion-infected CAD5 as well as N2A cells. Also, the intracellular localization of the retromer component VPS35 is different in prion-infected N2A cells when compared to non-infected cells. It has been reported that VPS35 levels are downregulated in CJD patient's brain. We found a similar decrease in VPS35 levels in 22L-CAD5 cells as well as in brains of mice

<footnote><sup>a</sup>Institute for Neurodegenerative Diseases, Weill Institute for Neurosciences, University of California, San Francisco, CA, USA;</footnote>

<footnote><sup>a</sup>Daiichi Sankyo Co., Ltd, Tokyo, Japan; <sup>c</sup>Department of Neurology, University of California, San Francisco, CA, USA</footnote>
<figure><img src="image_122.png" /></figure>

inoculated with 22L prions when compared to mock (PBS) inoculated controls at 125 dpi.

**Conclusion:** These findings indicate that prion infection dysregulates retromer mediated trafficking, potentially due to the decreased VPS35 levels. The dysregulation of intracellular vesicle trafficking could be a mechanism of neuronal damage caused by prion infection and represents a novel therapeutic target.

**212. Effects of E200K genetic predisposition to prion disease on cardiomyocyte cellular function, cytoskeleton structure, and protein expression**

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

CONTACT Aleksandar Wood aleksandar.wood@nih.gov

**Background/Introduction:** The prion protein (PrP) has been shown to have a role in cellular protection against oxidative stress in numerous cell types, including murine heart tissue. Likewise, cardiac autonomic impairment has been linked with prion disease. In genetic Creutzfeldt-Jakob Disease (CJD) caused by the E200K single amino acid mutation, intraneuronal PrP deposition is found in the brainstem, which may influence cardiovascular autonomic control. However, the direct functional influence of the E200K mutation on cardiomyocytes has not been investigated. We hypothesized that the E200K mutation may cause a loss of PrP functional capacity resulting in increased oxidative damage and deficient function.

**Materials and Methods:** Cardiomyocytes were differentiated from several human induced pluripotent stem cell lines. The underlying cells were either 'normal' (no predisposition to dementia), contained the E200K mutation within the prion gene (predisposed to developing genetic CJD), or trisomy 21 (causing Down's Syndrome in the donor). Baseline electrophysiological recordings were collected, measuring peak-to-peak amplitude as well as changes in overall channel activity over time. Experimental treatments were added and recordings were continued at specified time intervals, spanning from hours to days. Treatments were used to stimulate mitochondrial function, which would be expected to increase oxidative stress, and to inhibit contractility, reducing workload and therefore reducing oxidative stress. Twenty-four hours post-treatment, protein expression levels were assessed via Western Blot and Immunofluorescence.

**Results:** Preliminary electrophysiology recordings of cardiomyocyte cultures revealed that contractility of the cells varied basally and was altered by stimulating mitochondria. Expression levels of the superoxide dismutase (SOD) family was assessed as an indication of increased load from oxidative stressors; higher levels of expression can indicate compensatory efforts to negate the increased stress. E200K cardiomyocytes revealed increased SOD2 expression compared with controls, as well as increased prion protein (PrP) expression and increased B-Cell Lymphoma 2 (BCL2) expression – an anti-apoptotic marker.

**Conclusions:** The function, structure, and protein expression of cardiomyocytes was influenced by the E200K mutation. An impaired response to oxidative stress was indicated by changes in the localization and expression of SODs, PrP itself, and the anti-apoptotic protein, BCL2.

**KEYWORDS:** Prion; oxidative stress; reactive oxygen species; neurodegeneration; spongiform encephalopathy; cardiomyocyte; iPSC

**213. Prenatal noise stress aggravates cognitive decline and the onset and progression of β-amyloid pathology in a mouse model of Alzheimer's disease**

Zahra Jafari, Megan Okuma, Hadil Karem, Jogender Mehla, Bryan E. Kolb and Majid H. Mohajerani

Department of Neuroscience, Canadian Centre for Behavioural Neuroscience (CCBN), University of Lethbridge, Lethbridge, AB, Canada

CONTACT Zahra Jafari Zahra.jafari@uleth.ca

**ABSTRACT**

Environmental distresses occurring during the sensitive periods of early-life may exacerbate the vulnerability to develop physical and mental diseases in old age. Studies have shown the impact of prenatal stress (PS) on the endocrine development and reprogramming of hypothalamic-pituitary-adrenal (HPA) axis functions in association with cognitive development and susceptibility to neuropsychiatric disease. Long-term exposure to glucocorticoids can damage the brain and intensify the progression of Alzheimer's disease (AD)-like neuropathological changes, especially in females. There is, however, less information as to the link between PS and the risk of developing AD pathology during the lifespan. Male and female APP<sup>NL-G-F</sup>/NL-G-F offspring of dams exposed to gestational noise stress were compared with the control offspring in corticosterone

<footnote><sup>a</sup>National Institute of Allergy and Infectious Diseases (Laboratory of Persistent Viral Diseases); <sup>b</sup>Case Western Reserve University School of Medicine</footnote>
<figure><img src="image_126.png" /></figure>

inoculated with 22L prions when compared to mock (PBS) inoculated controls at 125 dpi.

**Conclusion:** These findings indicate that prion infection dysregulates retromer mediated trafficking, potentially due to the decreased VPS35 levels. The dysregulation of intracellular vesicle trafficking could be a mechanism of neuronal damage caused by prion infection and represents a novel therapeutic target.

**212. Effects of E200K genetic predisposition to prion disease on cardiomyocyte cellular function, cytoskeleton structure, and protein expression**

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

CONTACT Aleksandar Wood aleksandar.wood@nih.gov

**Background/Introduction:** The prion protein (PrP) has been shown to have a role in cellular protection against oxidative stress in numerous cell types, including murine heart tissue. Likewise, cardiac autonomic impairment has been linked with prion disease. In genetic Creutzfeldt-Jakob Disease (CJD) caused by the E200K single amino acid mutation, intraneuronal PrP deposition is found in the brainstem, which may influence cardiovascular autonomic control. However, the direct functional influence of the E200K mutation on cardiomyocytes has not been investigated. We hypothesized that the E200K mutation may cause a loss of PrP functional capacity resulting in increased oxidative damage and deficient function.

**Materials and Methods:** Cardiomyocytes were differentiated from several human induced pluripotent stem cell lines. The underlying cells were either 'normal' (no predisposition to dementia), contained the E200K mutation within the prion gene (predisposed to developing genetic CJD), or trisomy 21 (causing Down's Syndrome in the donor). Baseline electrophysiological recordings were collected, measuring peak-to-peak amplitude as well as changes in overall channel activity over time. Experimental treatments were added and recordings were continued at specified time intervals, spanning from hours to days. Treatments were used to stimulate mitochondrial function, which would be expected to increase oxidative stress, and to inhibit contractility, reducing workload and therefore reducing oxidative stress. Twenty-four hours post-treatment, protein expression levels were assessed via Western Blot and Immunofluorescence.

**Results:** Preliminary electrophysiology recordings of cardiomyocyte cultures revealed that contractility of the cells varied basally and was altered by stimulating mitochondria. Expression levels of the superoxide dismutase (SOD) family was assessed as an indication of increased load from oxidative stressors; higher levels of expression can indicate compensatory efforts to negate the increased stress. E200K cardiomyocytes revealed increased SOD2 expression compared with controls, as well as increased prion protein (PrP) expression and increased B-Cell Lymphoma 2 (BCL2) expression – an anti-apoptotic marker.

**Conclusions:** The function, structure, and protein expression of cardiomyocytes was influenced by the E200K mutation. An impaired response to oxidative stress was indicated by changes in the localization and expression of SODs, PrP itself, and the anti-apoptotic protein, BCL2.

**KEYWORDS:** Prion; oxidative stress; reactive oxygen species; neurodegeneration; spongiform encephalopathy; cardiomyocyte; iPSC

**213. Prenatal noise stress aggravates cognitive decline and the onset and progression of β-amyloid pathology in a mouse model of Alzheimer's disease**

Zahra Jafari, Megan Okuma, Hadil Karem, Jogender Mehla, Bryan E. Kolb and Majid H. Mohajerani

Department of Neuroscience, Canadian Centre for Behavioural Neuroscience (CCBN), University of Lethbridge, Lethbridge, AB, Canada

CONTACT Zahra Jafari Zahra.jafari@uleth.ca

**ABSTRACT**

Environmental distresses occurring during the sensitive periods of early-life may exacerbate the vulnerability to develop physical and mental diseases in old age. Studies have shown the impact of prenatal stress (PS) on the endocrine development and reprogramming of hypothalamic-pituitary-adrenal (HPA) axis functions in association with cognitive development and susceptibility to neuropsychiatric disease. Long-term exposure to glucocorticoids can damage the brain and intensify the progression of Alzheimer's disease (AD)-like neuropathological changes, especially in females. There is, however, less information as to the link between PS and the risk of developing AD pathology during the lifespan. Male and female APP<sup>NL-G-F</sup>/NL-G-F offspring of dams exposed to gestational noise stress were compared with the control offspring in corticosterone

<footnote><sup>a</sup>National Institute of Allergy and Infectious Diseases (Laboratory of Persistent Viral Diseases); <sup>b</sup>Case Western Reserve University School of Medicine</footnote>
<figure><img src="image_123.png" /></figure>

alternations, cognitive and motor performances, and the onset age and development of amyloid beta (Aβ) plaques across age. The hyperactivity of the HPA axis, spatial learning, and Aβ development were sex-specific showing persistent high levels of stress and further memory loss in females than males, especially in PS mice. The Aβ deposition was started earlier, by 2-3 months, and exhibited a heightened progression in PS animals. The PS also created a long-lasting anxiety-like behaviour and impairment in cognitive function and motor coordination. The findings suggest PS as a risk to exacerbate AD-like neuropathological changes during the lifespan, with higher susceptibility of females. The findings were discussed according to the most likely mechanisms for the PS effects, i.e., dysregulation of the neuroendocrine system and the placenta by the PS.

**KEYWORDS:** Prenatal stress; Alzheimer's disease; Aβ plaque; cognitive decline; motor impairment; prepulse inhibition; HPA axis

## 214. Identification of novel CWD strains

Debbie McKenzieª, Camilo Duque Velasquezª, Allen Herbstª, Elizabeth Triscottª, Jacques van der Merweª, Samia Hannaouiᵇ, Leonardo Cortezª, Sara Amidianª, Valerie Simª, Holger Willeª, Hermann Schatzlᵇ, Sabine Gilchᵇ and Judd Aikenª

CONTACT Debbie McKenzie Debbie.mckenzie@ualberta.ca

### ABSTRACT

Chronic wasting disease is a set of prion diseases infecting captive and wild cervids. As different CWD agents are transmitted between different cervid species and between the same species having different Prnp polymorphisms, novel CWD strains can be generated. These new strains can exhibit different biochemical properties as well as different conformers. Identification of novel CWD strains is critically important as the different strains may vary in their host ranges, increasing the potential for transmission to economically important species as well as zoonotic transmission.

A rate limiting step in the characterization of CWD strains is the identification of deer samples that potentially contain novel strains. Much of the strain generation likely occurs during early passage between different Prnp genotypes (of the same or different species). Traditionally these differences have been

identified following passages into rodent models (strains of lab mice, transgenic mice expressing different Prnp sequences and/or hamsters). Incubation periods can be long and the number of potential isolates is high making transmission experiments a slow, tedious, and expensive process. To streamline the process, we have identified a panel of *in vitro* analyses to help target samples for further characterization. For some isolates, potential new strains have been identified by western blot analysis, others by cervid cell assay, changes in the Prnp sequence, folding and/or aggregation differences as well as ability to seed reactions in RT-QuIC. Isolates of interest are then further characterized by transmission in a variety of different tg mouse lines, wild-type mice, hamsters, and voles. Using these criteria, five potential CWD strains have been identified.

## 215. Water quality improvement agent (new type CAC-717) also as prion disinfectant

Takashi Onoderaª, Koichi Furusakiᵇ, Makoto Haritaniª and Akikazu Sakudoᶜ

In Prion 2018 we reported electrically charged material (CAC-717) by continuously applying an electric field to mineral water containing calcium hydrogen carbonate. A Teflon insulation-coated electrostatic field electrode (N-800N, Mineral Activation Technical Research Center; Japan Patent No. 5864010) was used to create the electric field at a voltage of 2 × 10⁴ V for 48 h. CAC-717 solution in distilled water (Japan Patent No. 5778328, FDA/USA Regulation No. 880.6890 Class 1 disinfectant) had a pH of 12.39 ± 0.03 and contained calcium hydrogen carbonate particles (1,120 mg/L) and carbonate complex microparticles (50–500 nm) with a mesoscopic structure. CAC-717 works as a soil amendment was evaluated by growth of green tea leaves in Japanese farm lands. In Prion2019 we report that CAC-717 absorbed by ceramics were applied to fresh or sea water to work as water quality improvement agents.

Mouse prion (Chandler) infected N2a cell (ScN2a) lysate was subsequently mixed with equal volume of CAC-717 solution. As a negative control, uninfected N2a cell lysate was diluted 2-fold with PBS or RIPA buffer. 200 µg lysate protein was diluted in 100 µg of buffer (PBS or RIPA) and mixed with 100 µl of PBS. Mixtures were incubated at room temperature for 1 h then immediately diluted in PBS before determining the amount of prion in each sample for Western blotting with SAF83 monoclonal antibody. Prion was not detected in the ScN2a cell line lysate mixed with CAC-

<footnote>ªCentre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, AB, Canada; ᵇCalgary Prion Centre, University of Calgary, Calgary, AB, Canada</footnote>

<footnote>ªUniversity of Tokyo; ᵇMineral Activation Technical Research Center; ᶜUniversity of Ryukyus</footnote>
<figure><img src="image_127.png" /></figure>

alternations, cognitive and motor performances, and the onset age and development of amyloid beta (Aβ) plaques across age. The hyperactivity of the HPA axis, spatial learning, and Aβ development were sex-specific showing persistent high levels of stress and further memory loss in females than males, especially in PS mice. The Aβ deposition was started earlier, by 2-3 months, and exhibited a heightened progression in PS animals. The PS also created a long-lasting anxiety-like behaviour and impairment in cognitive function and motor coordination. The findings suggest PS as a risk to exacerbate AD-like neuropathological changes during the lifespan, with higher susceptibility of females. The findings were discussed according to the most likely mechanisms for the PS effects, i.e., dysregulation of the neuroendocrine system and the placenta by the PS.

**KEYWORDS:** Prenatal stress; Alzheimer's disease; Aβ plaque; cognitive decline; motor impairment; prepulse inhibition; HPA axis

## 214. Identification of novel CWD strains

Debbie McKenzieª, Camilo Duque Velasquezª, Allen Herbstª, Elizabeth Triscottª, Jacques van der Merweª, Samia Hannaouiᵇ, Leonardo Cortezª, Sara Amidianª, Valerie Simª, Holger Willeª, Hermann Schatzlᵇ, Sabine Gilchᵇ and Judd Aikenª

CONTACT Debbie McKenzie Debbie.mckenzie@ualberta.ca

## ABSTRACT

Chronic wasting disease is a set of prion diseases infecting captive and wild cervids. As different CWD agents are transmitted between different cervid species and between the same species having different Prnp polymorphisms, novel CWD strains can be generated. These new strains can exhibit different biochemical properties as well as different conformers. Identification of novel CWD strains is critically important as the different strains may vary in their host ranges, increasing the potential for transmission to economically important species as well as zoonotic transmission.

A rate limiting step in the characterization of CWD strains is the identification of deer samples that potentially contain novel strains. Much of the strain generation likely occurs during early passage between different Prnp genotypes (of the same or different species). Traditionally these differences have been

identified following passages into rodent models (strains of lab mice, transgenic mice expressing different Prnp sequences and/or hamsters). Incubation periods can be long and the number of potential isolates is high making transmission experiments a slow, tedious, and expensive process. To streamline the process, we have identified a panel of *in vitro* analyses to help target samples for further characterization. For some isolates, potential new strains have been identified by western blot analysis, others by cervid cell assay, changes in the Prnp sequence, folding and/or aggregation differences as well as ability to seed reactions in RT-QuIC. Isolates of interest are then further characterized by transmission in a variety of different tg mouse lines, wild-type mice, hamsters, and voles. Using these criteria, five potential CWD strains have been identified.

## 215. Water quality improvement agent (new type CAC-717) also as prion disinfectant

Takashi Onoderaª, Koichi Furusakiᵇ, Makoto Haritaniª and Akikazu Sakudoᶜ

In Prion 2018 we reported electrically charged material (CAC-717) by continuously applying an electric field to mineral water containing calcium hydrogen carbonate. A Teflon insulation-coated electrostatic field electrode (N-800N, Mineral Activation Technical Research Center; Japan Patent No. 5864010) was used to create the electric field at a voltage of 2 × 10⁴ V for 48 h. CAC-717 solution in distilled water (Japan Patent No. 5778328, FDA/USA Regulation No. 880.6890 Class 1 disinfectant) had a pH of 12.39 ± 0.03 and contained calcium hydrogen carbonate particles (1,120 mg/L) and carbonate complex microparticles (50–500 nm) with a mesoscopic structure. CAC-717 works as a soil amendment was evaluated by growth of green tea leaves in Japanese farm lands. In Prion2019 we report that CAC-717 absorbed by ceramics were applied to fresh or sea water to work as water quality improvement agents.

Mouse prion (Chandler) infected N2a cell (ScN2a) lysate was subsequently mixed with equal volume of CAC-717 solution. As a negative control, uninfected N2a cell lysate was diluted 2-fold with PBS or RIPA buffer. 200 µg lysate protein was diluted in 100 µg of buffer (PBS or RIPA) and mixed with 100 µl of PBS. Mixtures were incubated at room temperature for 1 h then immediately diluted in PBS before determining the amount of prion in each sample for Western blotting with SAF83 monoclonal antibody. Prion was not detected in the ScN2a cell line lysate mixed with CAC-

<footnote>ªCentre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, AB, Canada; ᵇCalgary Prion Centre, University of Calgary, Calgary, AB, Canada</footnote>

<footnote>ªUniversity of Tokyo; ᵇMineral Activation Technical Research Center; ᶜUniversity of Ryukyus</footnote>
<figure><img src="image_124.png" /></figure>

717 solution after testing with Western Blotting. However, $PrP^C$ levels was intact in both uninfected N2a cell and infected ScN2a cells, detected by Western Blotting.

CAC-717 ceramics placed inside concrete blocks were sprayed into sea water by the seashore of Shimane Prefecture, Japan. Then alteration of water environment was observed for 6 months. Number of sea urchin, sea cucumber, and abalones were calculated inside the concrete blocks with CAC-717 ceramics. Yield, as calculated by the number of these marine animals, was then determined. No animal was observed in the blocks with ceramics without CAC-717. While large amounts of animals were attracted and growing inside the blocks with CAC-717 ceramics.

Evaluation of water environment, nutrient content, composition, and microbiological activity tend to increase following use of water quality improvement agents. Numerous studies have therefore to be examined for the effect of water quality improvement agent, beside animal yield and quality. Our future aims are to determine the microbiological activity of CAC-717 ceramics after spraying with CAC-717.

## 216. Molecular dynamics study of PrP conversion

Lyudmyla Dorosh<sup>a,b</sup>, Min Wu<sup>a</sup>, Sandipan Chakraborty<sup>c</sup>, Holger Wille<sup>d,e</sup> and Maria Stepanova<sup>a,b</sup>

CONTACT Lyudmyla Dorosh dorosh@ualberta.ca

### ABSTRACT

Prion diseases are believed to be caused by a conversion of native cellular prion protein ($PrP^C$) into infectious β-rich aggregates ($PrP^{Sc}$). The structures of these aggregates and the molecular mechanisms of the conversion remain unknown, although numerous models for the structure of $PrP^{Sc}$ have been suggested [1]. In order to elucidate mechanisms of the conversion, we have done extensive all-atom molecular dynamics (MD) simulations tackling early stages of this process.

We employed several β-helical models of $PrP^{Sc}$. These include the recently proposed [2] 4-rung right-handed β-helical (RHBH) head-to-tail $moPrP^{Sc}$(89-230) model based on cryo-EM and X-ray fiber diffraction data and corrected with MD simulations; hypothetical 4-rung RHBH head-to-head and head-to-tail $moPrP^{Sc}$(89-230) models [3]; and a 4.5 rung left-

handed β-helical (LHBH) head-to-head $huPrP^{Sc}$(114-220) model built by computational threading of the PrP sequence onto published β-solenoid architectures. In simulations of the $PrP^C$ conversion, matching native $PrP^C$ constructs (solution NMR) were also included.

In our MD simulations of the above-mentioned $PrP^{Sc}$ models, the head-to-tail RHBH dimer [2] showed a higher stability in comparison to other constructs. However in different dimeric models, a preference for head-to-head configurations was found. A greater structural stability and an increase of β-content were also found in the dimers in comparison to monomers. The β-content and morphology were somewhat less stable in the LHBH model than in the RHBH models.

Simulations of the $PrP^C$ conversion containing native $PrP^C$ and $PrP^{Sc}$ revealed early unfolding events in $PrP^C$. Head-to-head contact of $PrP^C$ with a RHBH $PrP^{Sc}$ resulted in unfolding and formation of β-strands distant from the contact area, in regions of loop LH2H3 and helices H2 and H3, in first 100 ns. These changes remained stable for the next 150 ns. In systems with two $PrP^C$ molecules, similar trends towards partial unfolding of H2 and H3 with formation of β-strands were observed. During 155 ns simulations of native $PrP^C$ in a head-to-tail contact with monomeric RHBH $PrP^{Sc}$ [3], a pronouncedly better connection was observed. Distinct from other systems, the S1S2 bundle of $PrP^C$ approached the β-solenoid, partial unfolding of helix H1 occurred, and some of secondary structure around the loop LH2H3 was lost. However, our results suggest that fast conversion of $PrP^C$ has a low probability. The connection between S1, S2, and H2H3 bundle is not interrupted, stabilizing the remaining secondary structure in $PrP^C$.

### References

[1] Requena, Wille. Prion. 2014;8:60-66.

[2] Spagnolli et al., bioRxiv 2018; Preprint http://dx.doi.org/10.1101/505271.

[3] Requena, Chakraborty, personal communication.

## 217. The presence of autoantibodies against the prion protein is independent of PRNP mutations

Karl Frontzek<sup>a</sup>, Manfredi Carta<sup>a</sup>, Marco Losa<sup>a</sup>, Mirka Epskamp<sup>a</sup>, Georg Meisl<sup>b</sup>, Ulrike Camenisch<sup>c</sup>, Tuomas Knowles<sup>b</sup>, Simone Hornemann<sup>a</sup> and Adriano Aguzzi<sup>a</sup>; the THAUTAN-MC Investigators

<footnote><sup>a</sup>Department of Electrical and Computer Engineering, University of Alberta, Edmonton, Canada; <sup>b</sup>National Institute for Nanotechnology NRC; Edmonton, Canada; <sup>c</sup>Department of Microbiology, University of Calcutta, Kolkata, India; <sup>d</sup>Department of Biochemistry, University of Alberta, Edmonton, Canada; <sup>e</sup>Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Canada</footnote>

<footnote><sup>a</sup>Institute of Neuropathology, University of Zurich, Zurich, Switzerland; <sup>b</sup>Department of Chemistry, University of Cambridge, Cambridge, UK; <sup>c</sup>Institute of Surgical Pathology, University of Zurich, Zurich, Switzerland</footnote>
<figure><img src="image_128.png" /></figure>

717 solution after testing with Western Blotting. However, $PrP^C$ levels was intact in both uninfected N2a cell and infected ScN2a cells, detected by Western Blotting.

CAC-717 ceramics placed inside concrete blocks were sprayed into sea water by the seashore of Shimane Prefecture, Japan. Then alteration of water environment was observed for 6 months. Number of sea urchin, sea cucumber, and abalones were calculated inside the concrete blocks with CAC-717 ceramics. Yield, as calculated by the number of these marine animals, was then determined. No animal was observed in the blocks with ceramics without CAC-717. While large amounts of animals were attracted and growing inside the blocks with CAC-717 ceramics.

Evaluation of water environment, nutrient content, composition, and microbiological activity tend to increase following use of water quality improvement agents. Numerous studies have therefore to be examined for the effect of water quality improvement agent, beside animal yield and quality. Our future aims are to determine the microbiological activity of CAC-717 ceramics after spraying with CAC-717.

## 216. Molecular dynamics study of PrP conversion

Lyudmyla Dorosh<sup>a,b</sup>, Min Wu<sup>a</sup>, Sandipan Chakraborty<sup>c</sup>, Holger Wille<sup>d,e</sup> and Maria Stepanova<sup>a,b</sup>

<sup>a</sup>Department of Electrical and Computer Engineering, University of Alberta, Edmonton, Canada; <sup>b</sup>National Institute for Nanotechnology NRC; Edmonton, Canada; <sup>c</sup>Department of Microbiology, University of Calcutta, Kolkata, India; <sup>d</sup>Department of Biochemistry, University of Alberta, Edmonton, Canada; <sup>e</sup>Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Canada

CONTACT Lyudmyla Dorosh dorosh@ualberta.ca

### ABSTRACT

Prion diseases are believed to be caused by a conversion of native cellular prion protein ($PrP^C$) into infectious β-rich aggregates ($PrP^{Sc}$). The structures of these aggregates and the molecular mechanisms of the conversion remain unknown, although numerous models for the structure of $PrP^{Sc}$ have been suggested [1]. In order to elucidate mechanisms of the conversion, we have done extensive all-atom molecular dynamics (MD) simulations tackling early stages of this process.

We employed several β-helical models of $PrP^{Sc}$. These include the recently proposed [2] 4-rung right-handed β-helical (RHBH) head-to-tail $moPrP^{Sc}(89-230)$ model based on cryo-EM and X-ray fiber diffraction data and corrected with MD simulations; hypothetical 4-rung RHBH head-to-head and head-to-tail $moPrP^{Sc}(89-230)$ models [3]; and a 4.5 rung left-handed β-helical (LHBH) head-to-head $huPrP^{Sc}(114-220)$ model built by computational threading of the PrP sequence onto published β-solenoid architectures. In simulations of the $PrP^C$ conversion, matching native $PrP^C$ constructs (solution NMR) were also included.

In our MD simulations of the above-mentioned $PrP^{Sc}$ models, the head-to-tail RHBH dimer [2] showed a higher stability in comparison to other constructs. However in different dimeric models, a preference for head-to-head configurations was found. A greater structural stability and an increase of β-content were also found in the dimers in comparison to monomers. The β-content and morphology were somewhat less stable in the LHBH model than in the RHBH models.

Simulations of the $PrP^C$ conversion containing native $PrP^C$ and $PrP^{Sc}$ revealed early unfolding events in $PrP^C$. Head-to-head contact of $PrP^C$ with a RHBH $PrP^{Sc}$ resulted in unfolding and formation of β-strands distant from the contact area, in regions of loop LH2H3 and helices H2 and H3, in first 100 ns. These changes remained stable for the next 150 ns. In systems with two $PrP^C$ molecules, similar trends towards partial unfolding of H2 and H3 with formation of β-strands were observed. During 155 ns simulations of native $PrP^C$ in a head-to-tail contact with monomeric RHBH $PrP^{Sc}$ [3], a pronouncedly better connection was observed. Distinct from other systems, the S1S2 bundle of $PrP^C$ approached the β-solenoid, partial unfolding of helix H1 occurred, and some of secondary structure around the loop LH2H3 was lost. However, our results suggest that fast conversion of $PrP^C$ has a low probability. The connection between S1, S2, and H2H3 bundle is not interrupted, stabilizing the remaining secondary structure in $PrP^C$.

### References

[1] Requena, Wille. Prion. 2014;8:60-66.

[2] Spagnolli et al., bioRxiv 2018; Preprint http://dx.doi.org/10.1101/505271.

[3] Requena, Chakraborty, personal communication.

## 217. The presence of autoantibodies against the prion protein is independent of PRNP mutations

Karl Frontzek<sup>a</sup>, Manfredi Carta<sup>a</sup>, Marco Losa<sup>a</sup>, Mirka Epskamp<sup>a</sup>, Georg Meisl<sup>b</sup>, Ulrike Camenisch<sup>c</sup>, Tuomas Knowles<sup>b</sup>, Simone Hornemann<sup>a</sup> and Adriano Aguzzi<sup>a</sup>; the THAUTAN-MC Investigators

<footnote><sup>a</sup>Institute of Neuropathology, University of Zurich, Zurich, Switzerland; <sup>b</sup>Department of Chemistry, University of Cambridge, Cambridge, UK; <sup>c</sup>Institute of Surgical Pathology, University of Zurich, Zurich, Switzerland</footnote>
<figure><img src="image_125.png" /></figure>

## ABSTRACT

**Background:** Prion diseases are incurable diseases of the central nervous system, which not only occur as sporadic and infectious forms, but can also be transmitted through the germ line as autosomal dominant traits. What is more, genetic prion diseases usually manifest in late age, indicative of protective factors. Here we reasoned that subtle conformational alterations of pathogenic prion protein (PrP<sup>C</sup>) variants could stochastically generate immunogenic neo-epitopes, raising the question whether such protective factors may consist, at least in part, of anti-PrP<sup>C</sup> autoantibodies. We therefore conducted an extensive search for such autoantibodies in persons carrying pathogenic PRNP mutations and, for control, in their unaffected relatives.

**Methods:** In this case-control study, we have collected blood samples from individuals (n = 134) expressing a wide spectrum of disease-associated variants of the PRNP gene. Individuals with a positive family history of genetic prion disease (n = 79) but lacking disease-associated PRNP mutations served as controls. Antibody reactivity was measured using an indirect ELISA for the detection of human IgG<sub>1-4</sub> antibodies against full-length human prion protein. We evaluated the effects of age, gender, PRNP mutations, PRNP codon 129 polymorphism, clinical signs of prion disease, and total IgG levels on autoantibody reactivity. In a subset of patients, autoantibody reactivity was assessed longitudinally for up to 2 years after baseline measurements.

**Results:** We found that antibody reactivity was present in a subset of both PRNP mutation carriers and controls. Elder individuals were at lower odds for the presence of anti-PrP<sup>C</sup> autoantibodies, independently of total IgG levels or PRNP genotype. Gender, clinical signs of prion disease, pathogenic PRNP variants or p. 129 polymorphism did not influence autoantibody reactivity. Moreover, levels of anti-PrP<sup>C</sup> IgG were stable over up to 2 years.

**Conclusions:** This study shows that pathogenic PRNP variants do not notably stimulate antibody-mediated anti-PrP<sup>C</sup> immunity. Moreover, anti-PrP<sup>C</sup> IgG autoantibodies are independent of overt signs of prion disease. The presence of anti-PrP<sup>C</sup> autoantibodies in the general population without any disease-specific association suggests that relatively high titers of naturally occurring antibodies are tolerated. This observation suggests that such antibodies, if administered to humans, may not induce severe adverse reactions and may therefore serve as anti-prion therapeutics.

## 218. Single-molecule studies show α-synuclein aggregation is inhibited by anti-prion compounds and novel computationally-derived ligands

Chunhua Donga<sup>a</sup>, Lindsay Shearera<sup>a</sup>, Craig R. Garena<sup>a</sup>, Pascal Mercierb<sup>b</sup>, Nils O. Petersenc<sup>c</sup>, and Michael T. Woodsidea<sup>a</sup>

<sup>a</sup>Department of Physics, University of Alberta, Edmonton, AB, Canada; <sup>b</sup>NANUC National High Field Nuclear Magnetic Resonance Centre, Edmonton, AB, Canada; <sup>c</sup>Department of Chemistry, University of Alberta, Edmonton, AB, Canada; Edmonton, AB, Canada

## ABSTRACT

The intrinsically disordered protein α-synuclein (αS) is enriched in neurons and has been shown to be linked to Parkinson's disease (PD) and other related neurodegenerative disorders. Single αS molecules are postulated to undergo a misfolding/aggregation cascade that ultimately yields Lewy bodies, the histopathological hallmark of PD. The main neurotoxic species in this process have been shown experimentally to be prefibrillar oligomeric intermediates. We have investigated the inhibitory effects of various ligands on αS aggregation using single-molecule fluorescence cross correlation spectroscopy techniques and traditional ensemble ThT assays. These methods monitor respectively the influence of the ligands on the nucleation of small oligomers and the formation of fibrils. We characterized several inhibitors of αS aggregation from among known anti-prion agents and from a small library of compounds that were predicted to inhibit dimer formation based on computer simulations of αS dimer structures elucidated using single molecule force spectroscopy measurements of dimer aggregation. The ability of the same ligands to inhibit PrP aggregation and αS aggregation suggests there may be common features in the aggregation mechanisms that can be targeted for inhibition. The success of novel aggregation inhibitors discovered through computational simulations suggests that this approach provides a viable way to search for new inhibitors. Looking forward, we will broaden our search for additional novel candidates for development of PD therapeutics by testing αS with known inhibitors of other proteins that form amyloids (Aβ, tau, huntingtin, prion etc.), and by expanding the in silico discovery platform.

## References

[1] Spillantini et al. PNAS USA. 1998;95:6469-73.

[2] Masliah et al. Science. 2000;287:1265-69.

[3] Volles et al. Biochem. 2003;42:7871-8.
<figure><img src="image_129.png" /></figure>

## ABSTRACT

**Background:** Prion diseases are incurable diseases of the central nervous system, which not only occur as sporadic and infectious forms, but can also be transmitted through the germ line as autosomal dominant traits. What is more, genetic prion diseases usually manifest in late age, indicative of protective factors. Here we reasoned that subtle conformational alterations of pathogenic prion protein (PrP<sup>C</sup>) variants could stochastically generate immunogenic neo-epitopes, raising the question whether such protective factors may consist, at least in part, of anti-PrP<sup>C</sup> autoantibodies. We therefore conducted an extensive search for such autoantibodies in persons carrying pathogenic PRNP mutations and, for control, in their unaffected relatives.

**Methods:** In this case-control study, we have collected blood samples from individuals (n = 134) expressing a wide spectrum of disease-associated variants of the PRNP gene. Individuals with a positive family history of genetic prion disease (n = 79) but lacking disease-associated PRNP mutations served as controls. Antibody reactivity was measured using an indirect ELISA for the detection of human IgG<sub>1-4</sub> antibodies against full-length human prion protein. We evaluated the effects of age, gender, PRNP mutations, PRNP codon 129 polymorphism, clinical signs of prion disease, and total IgG levels on autoantibody reactivity. In a subset of patients, autoantibody reactivity was assessed longitudinally for up to 2 years after baseline measurements.

**Results:** We found that antibody reactivity was present in a subset of both PRNP mutation carriers and controls. Elder individuals were at lower odds for the presence of anti-PrP<sup>C</sup> autoantibodies, independently of total IgG levels or PRNP genotype. Gender, clinical signs of prion disease, pathogenic PRNP variants or p. 129 polymorphism did not influence autoantibody reactivity. Moreover, levels of anti-PrP<sup>C</sup> IgG were stable over up to 2 years.

**Conclusions:** This study shows that pathogenic PRNP variants do not notably stimulate antibody-mediated anti-PrP<sup>C</sup> immunity. Moreover, anti-PrP<sup>C</sup> IgG autoantibodies are independent of overt signs of prion disease. The presence of anti-PrP<sup>C</sup> autoantibodies in the general population without any disease-specific association suggests that relatively high titers of naturally occurring antibodies are tolerated. This observation suggests that such antibodies, if administered to humans, may not induce severe adverse reactions and may therefore serve as anti-prion therapeutics.

## 218. Single-molecule studies show α-synuclein aggregation is inhibited by anti-prion compounds and novel computationally-derived ligands

Chunhua Donga, Lindsay Shearera, Craig R. Garena, Pascal Mercierb, Nils O. Petersenc, and Michael T. Woodsidea

<sup>a</sup>Department of Physics, University of Alberta, Edmonton, AB, Canada; <sup>b</sup>NANUC National High Field Nuclear Magnetic Resonance Centre, Edmonton, AB, Canada; <sup>c</sup>Department of Chemistry, University of Alberta, Edmonton, AB, Canada; Edmonton, AB, Canada

## ABSTRACT

The intrinsically disordered protein α-synuclein (αS) is enriched in neurons and has been shown to be linked to Parkinson's disease (PD) and other related neurodegenerative disorders. Single αS molecules are postulated to undergo a misfolding/aggregation cascade that ultimately yields Lewy bodies, the histopathological hallmark of PD. The main neurotoxic species in this process have been shown experimentally to be prefibrillar oligomeric intermediates. We have investigated the inhibitory effects of various ligands on αS aggregation using single-molecule fluorescence cross correlation spectroscopy techniques and traditional ensemble ThT assays. These methods monitor respectively the influence of the ligands on the nucleation of small oligomers and the formation of fibrils. We characterized several inhibitors of αS aggregation from among known anti-prion agents and from a small library of compounds that were predicted to inhibit dimer formation based on computer simulations of αS dimer structures elucidated using single molecule force spectroscopy measurements of dimer aggregation. The ability of the same ligands to inhibit PrP aggregation and αS aggregation suggests there may be common features in the aggregation mechanisms that can be targeted for inhibition. The success of novel aggregation inhibitors discovered through computational simulations suggests that this approach provides a viable way to search for new inhibitors. Looking forward, we will broaden our search for additional novel candidates for development of PD therapeutics by testing αS with known inhibitors of other proteins that form amyloids (Aβ, tau, huntingtin, prion etc.), and by expanding the in silico discovery platform.

## References

[1] Spillantini et al. PNAS USA. 1998;95:6469-73.

[2] Masliah et al. Science. 2000;287:1265-69.

[3] Volles et al. Biochem. 2003;42:7871-8.
<figure><img src="image_126.png" /></figure>

[4] Schwille et al. Biophys. J. 1997;72:1878-86.

[5] Saeed. Et al. Am. J. Clin. Pathol. 1967;309:274-84.

[6] Priola et al. Science 2000;287:1503-6.

[7] Dee et al. Biochim. Biophys. Acta. 2012;1824:826-32.

## 219. Domain-specific quantification of PrP in cerebrospinal fluid by targeted mass spectrometry

Eric Vallabh Minikel<sup>a,b,c</sup>, Eric Kuhn<sup>d</sup>, Alexandra Cocco<sup>d</sup>, Sonia M Vallabh<sup>a,b,c</sup>, Chrissy Hartigan<sup>d</sup>, Andrew G Reidenbach<sup>a</sup>, Franc Llorens<sup>e,f</sup>, Inga Zerre<sup>e</sup>, Sabina Capellari<sup>g,h</sup>, Piero Parchi<sup>g,i</sup>, Stuart L. Schreiber<sup>a,j</sup>, Steven A. Carr<sup>d</sup>

<sup>a</sup>Center for the Science of Therapeutics, Broad Institute of MIT and Harvard, Cambridge, MA, USA; <sup>b</sup>Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, MA, USA; <sup>c</sup>Prion Alliance, Cambridge, MA, USA; <sup>d</sup>Proteomics Platform, Broad Institute of MIT and Harvard, Cambridge, MA, USA; <sup>e</sup>National Reference Center for TSE, Georg-August University, Göttingen, Germany; <sup>f</sup>Biomedical Research Networking Center on Neurodegenerative Diseases (CIBERNED), L'Hospitalet de Llobregat, Barcelona, NA, Spain; <sup>g</sup>IRCCS - Institute of Neurological Sciences, Bologna, Italy; <sup>h</sup>Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy; <sup>i</sup>Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy; <sup>j</sup>Department of Chemistry & Chemical Biology, Harvard University, Cambridge, MA, USA

CONTACT Eric Vallabh Minikel eminikel@broadinstitute.org

### ABSTRACT

Antisense oligonucleotides in preclinical development for prion disease will seek to lower PrP expression in the brain [1]. Trials of PrP-lowering therapies are likely to rely on quantification of PrP in cerebrospinal fluid (CSF) as a pharmacodynamic biomarker and possibly as a trial endpoint [1,2]. Studies using PrP ELISA kits have reproducibly shown that CSF PrP is lowered in the symptomatic phase of disease [2-6], a potential confounder for reading out the effect of PrP-lowering drugs in symptomatic patients. To date it has been unclear PrP's lowered abundance in CSF results from its incorporation into plaques [7], retention in intracellular compartments [8], downregulation as a function of the disease process [9], or other factors. Because misfolding or proteolytic cleavage could potentially render PrP invisible to ELISA even if its concentration were constant or increasing in disease, we sought to establish an orthogonal method for CSF PrP quantification. We developed a targeted mass spectrometry method based on multiple reaction monitoring (MRM) [10] of nine PrP tryptic peptides quantified relative to known concentrations of isotopically labelled standards. Analytical validation experiments showed technical replicate coefficients of variation below

15%, strong dilution linearity and spike recovery, and applicability to both CSF and brain homogenate and across humans as well as preclinical species of interest. In *N* = 55 CSF samples from individuals referred to prion surveillance centres with rapidly progressive dementia, all six human PrP peptides, spanning the N- and C-terminal domains of PrP, were uniformly reduced in prion disease cases compared to non-prion diagnoses, replicating the findings from ELISA studies. Our study provides evidence that lowered CSF PrP concentration in prion disease is a genuine result of the disease process and not merely an artefact of ELISA-based measurement, and we provide a novel method suitable for preclinical and clinical quantification of CSF PrP as a tool for drug development.

### References

[1] Vallabh, et al., In preparation.

[2] Vallabh, et al., bioRxiv 2018. DOI:10.1101/295063.

[3] Meyne, et al., J Alzheimers Dis. 2009;17:863.

[4] Dorey, et al., JAMA Neurol. 2015;72:267.

[5] Abu Rumeileh et al, J Alzheimers Dis. 2017;55:1471.

[6] Villar-Pique, Schmitz et al., Mol Neurobiol. 2018. DOI:10.1007/s12035-018-1251-1.

[7] Parchi et al., Ann Neurol. 1999;46:224

[8] Goold, et al., J Cell Sci. 2013, 126, 3552.

[9] Mays, et al., J Clin Invest. 2014;124:847.

[10] Carr, et al., Mol Cell Proteomics. 2014;13:907.

## 220. Light and electron microscopic studies of the 139A-H(Ha) strain of scrapie passed in hamsters

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

<sup>a</sup>Laboratory of Electron Microscopy and Neuropathology, Department of Molecular Pathology and Neuropathology, Chair of Oncology, Medical University Lodz, Poland; <sup>b</sup>Department of Extrapyramidal Diseases, Chair of Rehabilitation, Medical University of Lodz, Lodz, Poland; <sup>c</sup>Department of Anatomy and Neurobiology, Medical University of Gdansk, Gdansk, Poland

### ABSTRACT

We report here the light and electron microscopic neuropathology of the 139A-H strain of scrapie passed in Syrian golden hamsters.

**Material and methods:** This strain was derived from the Scrapie Sheep Brain Pool (SSBP)-1 lineage [3,5]. Briefly, scrapie from a natural source was passed through Cheviot sheep, Welsh Mountain sheep, goats, mice and led to the isolation of the 'Chandler' scrapie strain. The clone 139A strain was derived following passage in hamsters and this strain
<figure><img src="image_130.png" /></figure>

[4] Schwille et al. Biophys. J. 1997;72:1878-86.

[5] Saeed. Et al. Am. J. Clin. Pathol. 1967;309:274-84.

[6] Priola et al. Science 2000;287:1503-6.

[7] Dee et al. Biochim. Biophys. Acta. 2012;1824:826-32.

## 219. Domain-specific quantification of PrP in cerebrospinal fluid by targeted mass spectrometry

Eric Vallabh Minikel<sup>a,b,c</sup>, Eric Kuhn<sup>d</sup>, Alexandra Cocco<sup>d</sup>, Sonia M Vallabh<sup>a,b,c</sup>, Chrissy Hartigan<sup>d</sup>, Andrew G Reidenbach<sup>a</sup>, Franc Llorens<sup>e,f</sup>, Inga Zerre<sup>e</sup>, Sabina Capellari<sup>g,h</sup>, Piero Parchi<sup>g,i</sup>, Stuart L. Schreiber<sup>a,j</sup>, Steven A. Carr<sup>d</sup>

<sup>a</sup>Center for the Science of Therapeutics, Broad Institute of MIT and Harvard, Cambridge, MA, USA; <sup>b</sup>Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, MA, USA; <sup>c</sup>Prion Alliance, Cambridge, MA, USA; <sup>d</sup>Proteomics Platform, Broad Institute of MIT and Harvard, Cambridge, MA, USA; <sup>e</sup>National Reference Center for TSE, Georg-August University, Göttingen, Germany; <sup>f</sup>Biomedical Research Networking Center on Neurodegenerative Diseases (CIBERNED), L'Hospitalet de Llobregat, Barcelona, NA, Spain; <sup>g</sup>IRCCS - Institute of Neurological Sciences, Bologna, Italy; <sup>h</sup>Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy; <sup>i</sup>Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy; <sup>j</sup>Department of Chemistry & Chemical Biology, Harvard University, Cambridge, MA, USA

CONTACT Eric Vallabh Minikel eminikel@broadinstitute.org

### ABSTRACT

Antisense oligonucleotides in preclinical development for prion disease will seek to lower PrP expression in the brain [1]. Trials of PrP-lowering therapies are likely to rely on quantification of PrP in cerebrospinal fluid (CSF) as a pharmacodynamic biomarker and possibly as a trial endpoint [1,2]. Studies using PrP ELISA kits have reproducibly shown that CSF PrP is lowered in the symptomatic phase of disease [2-6], a potential confounder for reading out the effect of PrP-lowering drugs in symptomatic patients. To date it has been unclear PrP's lowered abundance in CSF results from its incorporation into plaques [7], retention in intracellular compartments [8], downregulation as a function of the disease process [9], or other factors. Because misfolding or proteolytic cleavage could potentially render PrP invisible to ELISA even if its concentration were constant or increasing in disease, we sought to establish an orthogonal method for CSF PrP quantification. We developed a targeted mass spectrometry method based on multiple reaction monitoring (MRM) [10] of nine PrP tryptic peptides quantified relative to known concentrations of isotopically labelled standards. Analytical validation experiments showed technical replicate coefficients of variation below

15%, strong dilution linearity and spike recovery, and applicability to both CSF and brain homogenate and across humans as well as preclinical species of interest. In N = 55 CSF samples from individuals referred to prion surveillance centres with rapidly progressive dementia, all six human PrP peptides, spanning the N- and C-terminal domains of PrP, were uniformly reduced in prion disease cases compared to non-prion diagnoses, replicating the findings from ELISA studies. Our study provides evidence that lowered CSF PrP concentration in prion disease is a genuine result of the disease process and not merely an artefact of ELISA-based measurement, and we provide a novel method suitable for preclinical and clinical quantification of CSF PrP as a tool for drug development.

### References

[1] Vallabh, et al., In preparation.

[2] Vallabh, et al., bioRxiv 2018. DOI:10.1101/295063.

[3] Meyne, et al., J Alzheimers Dis. 2009;17:863.

[4] Dorey, et al., JAMA Neurol. 2015;72:267.

[5] Abu Rumeileh et al, J Alzheimers Dis. 2017;55:1471.

[6] Villar-Pique, Schmitz et al., Mol Neurobiol. 2018. DOI:10.1007/s12035-018-1251-1.

[7] Parchi et al., Ann Neurol. 1999;46:224.

[8] Goold, et al., J Cell Sci. 2013, 126, 3552.

[9] Mays, et al., J Clin Invest. 2014;124:847.

[10] Carr, et al., Mol Cell Proteomics. 2014;13:907.

## 220. Light and electron microscopic studies of the 139A-H(Ha) strain of scrapie passed in hamsters

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

<sup>a</sup>Laboratory of Electron Microscopy and Neuropathology, Department of Molecular Pathology and Neuropathology, Chair of Oncology, Medical University Lodz, Poland; <sup>b</sup>Department of Extrapyramidal Diseases, Chair of Rehabilitation, Medical University of Lodz, Lodz, Poland; <sup>c</sup>Department of Anatomy and Neurobiology, Medical University of Gdansk, Gdansk, Poland

### ABSTRACT

We report here the light and electron microscopic neuropathology of the 139A-H strain of scrapie passed in Syrian golden hamsters.

**Material and methods:** This strain was derived from the Scrapie Sheep Brain Pool (SSBP)-1 lineage [3,5]. Briefly, scrapie from a natural source was passed through Cheviot sheep, Welsh Mountain sheep, goats, mice and led to the isolation of the 'Chandler' scrapie strain. The clone 139A strain was derived following passage in hamsters and this strain
was made available for us by Dr. Richard I Carp, IBR, Staten Island, USA.

**Analysis of PrP immunostaining:** The intensity of the immunostaining were quantified in a single hamster brain using the images obtained from the AxioScan. Z1 system (Zeiss, Germany) using the Zeiss Zen 2.3 (Blue Edition) Software.

**Results:** The general neuropathological picture consisted of spongiform change and severe astrocytic gliosis. The topography of PrP was variable, the highest signal was observed in the CA2-molecular layer, CA1-pyramidal and entorhinal cortex. Double immunofluorescence stainings showed dot-like immunoreactivity of microtubule associated protein (LC3) in the cell bodies while axons more often showed fibrillar pattern. Interestingly, some neuronal processes showed only neurofilament protein and LC3 positive structures were depleted of NFP immunoreactivity. The electron microscopy consisted of:

(1) Spongiform vacuoles – these are always membrane bound and contained secondary vacuoles (i.e. membrane-bound compartments or vesicles within vacuoles) and curled membraned fragments.

(2) Tubulovesicular structures (TVS) – these are vesicular structures of approximate 27 nm in diameter within neuronal processes – i.e. axonal terminal or dendrites. TVS are smaller and of higher electron density than synaptic vesicles. The significance of TVS remains unknown.

(3) Dystrophic neurites. Dendrites or axonal terminals and terminals filled with electron-dense bodies, including small autophagic vacuoles.

(4) Apoptotic cell nuclei.

(5) ‘Whorls’, a concentric arrays of membranes were visible. A significance of those structures is unknown.

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

**KEYWORDS:** Scrapie; electron microscopy; topography of lesion

**221. Purification of small, non-fibrillar and infectious prion particles from the brain of patients with GSS-A117V**

Ilaria Vanniª, Laura Pirisinuª, Claudia Y. Acevedo-Morantesᵇ, Michele A. Di Bariª, Claudia D'Agostinoª, Stefano Marconª, Geraldina Riccardiª, Elena Espositoª,

Pierluigi Gambettiᶜ, Umberto Agrimiª, Holger Willeᵇ and Romolo Nonnoª

ªIstituto Superiore di Sanità, Department of Food Safety, Nutrition and Veterinary Public Health, Rome, Italy; ᵇCentre for Prions and Protein Folding Diseases & Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada; ᶜDepartment of Pathology, Case Western Reserve University, Cleveland, Ohio, USA

CONTACT Ilaria Vanni ilaria.vanni@iss.it

**ABSTRACT**

Gerstmann-Sträussler-Scheinker disease (GSS) is an inherited prion disease associated with mutations in the PRNP gene. We recently reported that GSS with the A117V mutation, characterized by $PrP^{Sc}$ with a protease resistant core made up of N- and C-terminally cleaved PrP peptides of ~7 kDa (7 kDa $PrP^{res}$), is transmissible in bank voles. However, definite proof of the infectious nature of this truncated $PrP^{Sc}$ type is still missing. We thus attempted to evaluate the GSS-A117V 7 kDa $PrP^{res}$ role in terms of infectivity, strain characteristics, and structural features.

At first, we determined if the infectivity of GSS-A117V $PrP^{Sc}$ is resistant to PK, by parallel end-point titrations in voles of PK-treated or untreated brain homogenates. Unexpectedly, GSS-A117V showed an extremely high infectious titer ($10^{9.3}$ ID$_{50}$ U/g), which was not significantly affected by PK treatment ($10^{9.0}$ ID$_{50}$ U/g). We then aimed at purifying the PK-resistant GSS-A117V prions by using a recently published protocol¹ in order to determine the amount of infectivity and $PrP^{res}$ in the different fractions, alongside with the morphological characteristics of purified $PrP^{res}$ aggregates by EM.

Purified pellet fractions from GSS-A117V contained the expected N- and C-terminally cleaved 7 kDa $PrP^{res}$, although the yield of $PrP^{res}$ was lower than that of a scrapie sample used as a control. We found that this lower yield depended on the low density/small size of GSS-A117V $PrP^{res}$, as it was mainly retained in the last supernatant fraction. All fractions were highly infectious, thus confirming the infectious nature of the 7 kDa $PrP^{res}$, with infectivity levels that directly correlated with the $PrP^{res}$ amount detected. Finally, EM analysis of these fractions showed no presence of amyloid fibrils, only very small and indistinct, non-fibrillar $PrP^{Sc}$ particles were detected. In contrast, $PrP^{Sc}$ fibrils with the expected morphology were recovered from the scrapie control sample.

Our study demonstrates that purified aggregates of 7 kDa $PrP^{res}$ are fully infectious and encode the biochemical and biological strain features of the original sample, implying that the C-terminus of $PrP^{Sc}$ is dispensable for infectivity and strain features. Finally, the non-fibrillar morphology of these unglycosylated and non-GPI-anchored, small and indistinct prion particles, coupled
<figure><img src="image_131.png" /></figure>

was made available for us by Dr. Richard I Carp, IBR, Staten Island, USA.

**Analysis of PrP immunostaining:** The intensity of the immunostaining were quantified in a single hamster brain using the images obtained from the AxioScan. Z1 system (Zeiss, Germany) using the Zeiss Zen 2.3 (Blue Edition) Software.

**Results:** The general neuropathological picture consisted of spongiform change and severe astrocytic gliosis. The topography of PrP was variable, the highest signal was observed in the CA2-molecular layer, CA1-pyramidal and entorhinal cortex. Double immunofluorescence stainings showed dot-like immunoreactivity of microtubule associated protein (LC3) in the cell bodies while axons more often showed fibrillar pattern. Interestingly, some neuronal processes showed only neurofilament protein and LC3 positive structures were depleted of NFP immunoreactivity. The electron microscopy consisted of:

(1) Spongiform vacuoles – these are always membrane bound and contained secondary vacuoles (i.e. membrane-bound compartments or vesicles within vacuoles) and curled membraned fragments.

(2) Tubulovesicular structures (TVS) – these are vesicular structures of approximate 27 nm in diameter within neuronal processes – i.e. axonal terminal or dendrites. TVS are smaller and of higher electron density than synaptic vesicles. The significance of TVS remains unknown.

(3) Dystrophic neurites. Dendrites or axonal terminals and terminals filled with electron-dense bodies, including small autophagic vacuoles.

(4) Apoptotic cell nuclei.

(5) ‘Whorls’, a concentric arrays of membranes were visible. A significance of those structures is unknown.

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

**KEYWORDS:** Scrapie; electron microscopy; topography of lesion

**221. Purification of small, non-fibrillar and infectious prion particles from the brain of patients with GSS-A117V**

Ilaria Vanniª, Laura Pirisinuª, Claudia Y. Acevedo-Morantesᵇ, Michele A. Di Bariª, Claudia D'Agostinoª, Stefano Marconª, Geraldina Riccardiª, Elena Espositoª,

Pierluigi Gambettiᶜ, Umberto Agrimiª, Holger Willeᵇ and Romolo Nonnoª

ªIstituto Superiore di Sanità, Department of Food Safety, Nutrition and Veterinary Public Health, Rome, Italy; ᵇCentre for Prions and Protein Folding Diseases & Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada; ᶜDepartment of Pathology, Case Western Reserve University, Cleveland, Ohio, USA

CONTACT Ilaria Vanni ilaria.vanni@iss.it

**ABSTRACT**

Gerstmann-Sträussler-Scheinker disease (GSS) is an inherited prion disease associated with mutations in the PRNP gene. We recently reported that GSS with the A117V mutation, characterized by $PrP^{Sc}$ with a protease resistant core made up of N- and C-terminally cleaved PrP peptides of ~7 kDa (7 kDa $PrP^{res}$), is transmissible in bank voles. However, definite proof of the infectious nature of this truncated $PrP^{Sc}$ type is still missing. We thus attempted to evaluate the GSS-A117V 7 kDa $PrP^{res}$ role in terms of infectivity, strain characteristics, and structural features.

At first, we determined if the infectivity of GSS-A117V $PrP^{Sc}$ is resistant to PK, by parallel end-point titrations in voles of PK-treated or untreated brain homogenates. Unexpectedly, GSS-A117V showed an extremely high infectious titer ($10^{9.3}$ ID$_{50}$ U/g), which was not significantly affected by PK treatment ($10^{9.0}$ ID$_{50}$ U/g). We then aimed at purifying the PK-resistant GSS-A117V prions by using a recently published protocol¹ in order to determine the amount of infectivity and $PrP^{res}$ in the different fractions, alongside with the morphological characteristics of purified $PrP^{res}$ aggregates by EM.

Purified pellet fractions from GSS-A117V contained the expected N- and C-terminally cleaved 7 kDa $PrP^{res}$, although the yield of $PrP^{res}$ was lower than that of a scrapie sample used as a control. We found that this lower yield depended on the low density/small size of GSS-A117V $PrP^{res}$, as it was mainly retained in the last supernatant fraction. All fractions were highly infectious, thus confirming the infectious nature of the 7 kDa $PrP^{res}$, with infectivity levels that directly correlated with the $PrP^{res}$ amount detected. Finally, EM analysis of these fractions showed no presence of amyloid fibrils, only very small and indistinct, non-fibrillar $PrP^{Sc}$ particles were detected. In contrast, $PrP^{Sc}$ fibrils with the expected morphology were recovered from the scrapie control sample.

Our study demonstrates that purified aggregates of 7 kDa $PrP^{res}$ are fully infectious and encode the biochemical and biological strain features of the original sample, implying that the C-terminus of $PrP^{Sc}$ is dispensable for infectivity and strain features. Finally, the non-fibrillar morphology of these unglycosylated and non-GPI-anchored, small and indistinct prion particles, coupled
<figure><img src="image_127.png" /></figure>

to the very high levels of infectivity detected in GSS-A117V, is reminiscent of previous data on non-fibrillar prion particles as the most efficient initiators of TSE diseases [2]. Our findings provide new molecular and morphological constraints on the structure of infectious prions.

## References

[1] Wenborn, et al., 2015.

[2] Silveira, et al., 2005.

## 222. The impact of cellular prion protein expression levels in 5xFAD mouse behaviour and lifespan

Angela Silva-Correiaª, Matthias Schmitzᵇ and Inga Zerrᶜ

ªMedical University of Göttingen; ᵇGeorg-August University; ᶜNational TSE Reference Center, Göttingen, Germany

## ABSTRACT

Alzheimer's disease is the most prevalent dementia causing progressive impairment of cognition, mood, speech, task performance ability, behaviour, and memory. Since all treatments for AD are primarily symptomatic, the development of disease modifying approaches continues to be an area of intense focus.

Previous studies already indicated an important role of the cellular prion protein (PrPC) as a regulator of the A β mediated toxicity by acting as a potential receptor. In this project we intended to evaluate different expression levels of PrPC and its impact of amyloid pathology in our animal model. We had already established three different bi-transgenic mouse line 5xFADPrnp⁺/⁺, 5xFADPrnp⁺ and 5xFADPrnp⁻/⁻ by crossing of PrPC knockout (Prnp⁻/⁻) mice with 5xFADPrnp⁺/⁺ mice (Tg6799) exhibiting mutations in the APP and presenilin genes. We observed a direct influence of PrPC expression levels on A β -induced behaviour deficits in our mice lines. 5xFADPrnp⁻/⁻ mice showed the biggest delay of behaviour impairment (cognition, anxiety or nest building) and 5xFADPrnp⁺/⁺ mice showed the fastest onset of behaviour impairment. Since a PrPC-knockout cannot prevent A β -induced deficits, we propose that further proteins and co-factor are involved in the uptake of A β and the accompanied cellular dysfunctions.

## 223. Scrapie in white-tailed deer: a strain of the CWD agent that efficiently transmits to sheep?

Justin J. Greenleeª, Robyn D. Kokemullerª, S. Jo Mooreª and Heather West Greenleeᵇ

ªVirus and Prion Research Unit, National Animal Disease Center, USDA, Agricultural Research Service, Ames, IA, USA; ᵇDepartment of Biomedical Sciences, Iowa State University College of Veterinary Medicine, Ames, IA, USA

CONTACT Justin J. Greenlee Justin.Greenlee@ars.usda.gov

## ABSTRACT

Scrapie is a transmissible spongiform encephalopathy of sheep and goats that is associated with widespread accumulation of abnormal prion protein (PrP<sup>Sc</sup>) in the central nervous and lymphoid tissues. Chronic wasting disease (CWD) is the natural prion disease of cervid species, and the tissue distribution of PrP<sup>Sc</sup> in affected cervids is similar to scrapie in sheep. There are several lines of evidence that suggest that multiple strains of CWD exist, which may affect the agent's potential to transmit to hosts of the same or different species. We inoculated white-tailed deer with the scrapie agent from ARQ/ARQ sheep, which resulted in 100% attack rates by either the intracranial or oronasal route of inoculation. When examining tissues from the brainstems or lymphoid tissues by traditional diagnostic methods such as immunohistochemistry or western blots, it is difficult to differentiate tissues from deer infected with scrapie from those infected with CWD. However, there are several important differences between tissues from scrapie-infected white-tailed deer (WTD scrapie) and those infected with CWD (WTD CWD). First, there are different patterns of PrP<sup>Sc</sup> deposition in the brains of infected deer: brain tissues from deer with WTD scrapie had predominantly particulate and stellate immunoreactivity whereas those from deer with WTD-CWD had large aggregates and plaque-like deposits. Secondly, the incubation periods of WTD scrapie isolates are longer than CWD isolates in mice expressing cervid prion protein. Most notably, the transmission potential of these two isolates back to sheep is distinctly different. Attempts to transmit various CWD isolates to sheep by the oral or oronasal routes have been unsuccessful despite observation periods of up to 7 years. However, WTD scrapie efficiently transmitted back to sheep by the oronasal route. Upon transmission back to sheep, the WTD scrapie isolate exhibited different phenotypic properties when compared to the sheep receiving the original sheep scrapie inoculum including different genotype susceptibilities, distinct PrP<sup>Sc</sup> deposition patterns, and much more rapid incubation periods in transgenic mice expressing the ovine prion protein. The scrapie agent readily transmits between sheep and deer after oronasal exposure. This could confound the identification of CWD strains in deer and the eradication of scrapie from sheep.
<figure><img src="image_132.png" /></figure>

to the very high levels of infectivity detected in GSS-A117V, is reminiscent of previous data on non-fibrillar prion particles as the most efficient initiators of TSE diseases [2]. Our findings provide new molecular and morphological constraints on the structure of infectious prions.

## References

[1] Wenborn, et al., 2015.

[2] Silveira, et al., 2005.

## 222. The impact of cellular prion protein expression levels in 5xFAD mouse behaviour and lifespan

Angela Silva-Correiaª, Matthias Schmitzᵇ and Inga Zerrᶜ

ªMedical University of Göttingen; ᵇGeorg-August University; ᶜNational TSE Reference Center, Göttingen, Germany

## ABSTRACT

Alzheimer's disease is the most prevalent dementia causing progressive impairment of cognition, mood, speech, task performance ability, behaviour, and memory. Since all treatments for AD are primarily symptomatic, the development of disease modifying approaches continues to be an area of intense focus.

Previous studies already indicated an important role of the cellular prion protein (PrPC) as a regulator of the A β mediated toxicity by acting as a potential receptor. In this project we intended to evaluate different expression levels of PrPC and its impact of amyloid pathology in our animal model. We had already established three different bi-transgenic mouse line 5xFADPrnp⁺/⁺, 5xFADPrnp⁺ and 5xFADPrnp⁻/⁻ by crossing of PrPC knockout (Prnp⁻/⁻) mice with 5xFADPrnp⁺/⁺ mice (Tg6799) exhibiting mutations in the APP and presenilin genes. We observed a direct influence of PrPC expression levels on A β -induced behaviour deficits in our mice lines. 5xFADPrnp⁻/⁻ mice showed the biggest delay of behaviour impairment (cognition, anxiety or nest building) and 5xFADPrnp⁺/⁺ mice showed the fastest onset of behaviour impairment. Since a PrPC-knockout cannot prevent A β -induced deficits, we propose that further proteins and co-factor are involved in the uptake of A β and the accompanied cellular dysfunctions.

## 223. Scrapie in white-tailed deer: a strain of the CWD agent that efficiently transmits to sheep?

Justin J. Greenleeª, Robyn D. Kokemullerª, S. Jo Mooreª and Heather West Greenleeᵇ

ªVirus and Prion Research Unit, National Animal Disease Center, USDA, Agricultural Research Service, Ames, IA, USA; ᵇDepartment of Biomedical Sciences, Iowa State University College of Veterinary Medicine, Ames, IA, USA

CONTACT Justin J. Greenlee Justin.Greenlee@ars.usda.gov

## ABSTRACT

Scrapie is a transmissible spongiform encephalopathy of sheep and goats that is associated with widespread accumulation of abnormal prion protein (PrP<sup>Sc</sup>) in the central nervous and lymphoid tissues. Chronic wasting disease (CWD) is the natural prion disease of cervid species, and the tissue distribution of PrP<sup>Sc</sup> in affected cervids is similar to scrapie in sheep. There are several lines of evidence that suggest that multiple strains of CWD exist, which may affect the agent's potential to transmit to hosts of the same or different species. We inoculated white-tailed deer with the scrapie agent from ARQ/ARQ sheep, which resulted in 100% attack rates by either the intracranial or oronasal route of inoculation. When examining tissues from the brainstems or lymphoid tissues by traditional diagnostic methods such as immunohistochemistry or western blots, it is difficult to differentiate tissues from deer infected with scrapie from those infected with CWD. However, there are several important differences between tissues from scrapie-infected white-tailed deer (WTD scrapie) and those infected with CWD (WTD CWD). First, there are different patterns of PrP<sup>Sc</sup> deposition in the brains of infected deer: brain tissues from deer with WTD scrapie had predominantly particulate and stellate immunoreactivity whereas those from deer with WTD-CWD had large aggregates and plaque-like deposits. Secondly, the incubation periods of WTD scrapie isolates are longer than CWD isolates in mice expressing cervid prion protein. Most notably, the transmission potential of these two isolates back to sheep is distinctly different. Attempts to transmit various CWD isolates to sheep by the oral or oronasal routes have been unsuccessful despite observation periods of up to 7 years. However, WTD scrapie efficiently transmitted back to sheep by the oronasal route. Upon transmission back to sheep, the WTD scrapie isolate exhibited different phenotypic properties when compared to the sheep receiving the original sheep scrapie inoculum including different genotype susceptibilities, distinct PrP<sup>Sc</sup> deposition patterns, and much more rapid incubation periods in transgenic mice expressing the ovine prion protein. The scrapie agent readily transmits between sheep and deer after oronasal exposure. This could confound the identification of CWD strains in deer and the eradication of scrapie from sheep.
<figure><img src="image_128.png" /></figure>

**224. NMR fragment screening to discover low molecular weight prion protein binders**

Andrew Reidenbach, Eric Minikel, Sonia Vallabh, Alison Leed, Jenna Yehl, Jayme Dahlin, Florence Wagner, Chris Lemke, Colin Garvie, Virendar Kaushik, Mike Mesleh and Stuart Schreiber

Broad Institute of MIT and Harvard

CONTACT Andrew Reidenbach areidenb@broadinstitute.org

**ABSTRACT**

Prion disease is a rapidly progressive neurodegenerative disorder caused by misfolding and aggregation of the prion protein (PrP<sup>C</sup>), and there are currently no therapeutic options. Attempts to target and disrupt misfolded PrP (PrP<sup>Sc</sup>) have resulted in emergent drug resistance and molecules that lack efficacy on human prion strains. Ligands of PrP<sup>C</sup> could antagonize prion formation by stabilizing the native protein or by targeting it for degradation, but no validated small-molecule binders have been discovered to date. We screened 6631 low molecular weight fragments for recombinant human PrP binding using <sup>19</sup>F and saturation transfer difference (STD) NMR. 80 actives were re-tested using protein-observed transverse relaxation optimized spectroscopy (TROSY), an orthogonal 2D NMR method. A single benzimidazole derivative validated in concentration-response and its analogues exhibited a structure-activity relationship, albeit with weak affinity (K<sub>d</sub> >1 mM). The exceptionally low hit rate and weak affinity observed here suggest that PrP is a difficult target for small molecule binders. Nevertheless, our studies may provide a starting point for structural biology and medicinal chemistry efforts to identify more potent ligands through structure-based drug design.

**KEYWORDS:** Fragment screening; NMR; binders

**225. Public acceptability of chronic wasting disease management approaches in Canada: a paired comparison approach**

Geoffrey Durocher, Marty (M.K.) Luckert, Ellen Goddard and Vic (W.L.) Adamowicz

Department of Resource Economics and Environmental Sociology, University of Alberta, Edmonton, Alberta, Canada

CONTACT Geoffrey Durocher durocher@ualberta.ca

**ABSTRACT**

**Background:** Chronic Wasting Disease (CWD) is a prion disease found among both captive and wild populations of cervids and its prevalence has been increasing over time. As CWD spreads, a number of stakeholders are affected, including hunters, landowners, and the general public. When deciding upon management options for CWD, it is important to consider the preferences of the stakeholders involved. The goal of this research is to investigate the acceptability of CWD management approaches through the eyes of stakeholders.

**Methods:** We employ a paired comparison approach, where respondents indicate their preferences across a series of proposed pairs of CWD management options. Management options were developed based on practices that have been used in CWD-endemic areas and based on ideas expressed by focus groups that we conducted with decision-makers representing various stakeholder interests. We develop separate series of management options for the general public, hunters, and landowners. Responses were collected from approximately 5200 individuals across Canada using an internet-based survey and a sample from an online panel. Respondents were asked to choose between pairs of management options, while considering their preferences associated with the alternative approaches. After each pair of options, respondents were asked whether they preferred their chosen option, or no action be taken. Preferences were analysed using conditional logistic regressions-based on random utility models.

**Results:** Little heterogeneity existed in the preferences of different stakeholders from different demographic groups. For the most part, reducing deer numbers in high risk CWD areas through the increased use of hunters was preferred to using government sharpshooters. Likewise, restricting the movement of carcasses and hunted products was also generally positively received by most groups, with the notable exception of hunters. There were also options that were almost universally disliked. For example, increasing incentives for private landowners to allow hunting on their lands, by allowing them to charge hunters for access, was even disliked by landowners. We also find that most respondents prefer some action to inaction.

**Conclusions:** As CWD increases in spread and prevalence, policymakers are deciding which management options should be pursued. Our results provide policymakers with important information regarding
**224. NMR fragment screening to discover low molecular weight prion protein binders**

Andrew Reidenbach, Eric Minikel, Sonia Vallabh, Alison Leed, Jenna Yehl, Jayme Dahlin, Florence Wagner, Chris Lemke, Colin Garvie, Virendar Kaushik, Mike Mesleh and Stuart Schreiber

Broad Institute of MIT and Harvard

CONTACT Andrew Reidenbach areidenb@broadinstitute.org

**ABSTRACT**

Prion disease is a rapidly progressive neurodegenerative disorder caused by misfolding and aggregation of the prion protein (PrP<sup>C</sup>), and there are currently no therapeutic options. Attempts to target and disrupt misfolded PrP (PrP<sup>Sc</sup>) have resulted in emergent drug resistance and molecules that lack efficacy on human prion strains. Ligands of PrP<sup>C</sup> could antagonize prion formation by stabilizing the native protein or by targeting it for degradation, but no validated small-molecule binders have been discovered to date. We screened 6631 low molecular weight fragments for recombinant human PrP binding using <sup>19</sup>F and saturation transfer difference (STD) NMR. 80 actives were re-tested using protein-observed transverse relaxation optimized spectroscopy (TROSY), an orthogonal 2D NMR method. A single benzimidazole derivative validated in concentration-response and its analogues exhibited a structure-activity relationship, albeit with weak affinity (K<sub>d</sub> >1 mM). The exceptionally low hit rate and weak affinity observed here suggest that PrP is a difficult target for small molecule binders. Nevertheless, our studies may provide a starting point for structural biology and medicinal chemistry efforts to identify more potent ligands through structure-based drug design.

**KEYWORDS:** Fragment screening; NMR; binders

**225. Public acceptability of chronic wasting disease management approaches in Canada: a paired comparison approach**

Geoffrey Durocher, Marty (M.K.) Luckert, Ellen Goddard and Vic (W.L.) Adamowicz

Department of Resource Economics and Environmental Sociology, University of Alberta, Edmonton, Alberta, Canada

CONTACT Geoffrey Durocher durocher@ualberta.ca

**ABSTRACT**

**Background:** Chronic Wasting Disease (CWD) is a prion disease found among both captive and wild populations of cervids and its prevalence has been increasing over time. As CWD spreads, a number of stakeholders are affected, including hunters, landowners, and the general public. When deciding upon management options for CWD, it is important to consider the preferences of the stakeholders involved. The goal of this research is to investigate the acceptability of CWD management approaches through the eyes of stakeholders.

**Methods:** We employ a paired comparison approach, where respondents indicate their preferences across a series of proposed pairs of CWD management options. Management options were developed based on practices that have been used in CWD-endemic areas and based on ideas expressed by focus groups that we conducted with decision-makers representing various stakeholder interests. We develop separate series of management options for the general public, hunters, and landowners. Responses were collected from approximately 5200 individuals across Canada using an internet-based survey and a sample from an online panel. Respondents were asked to choose between pairs of management options, while considering their preferences associated with the alternative approaches. After each pair of options, respondents were asked whether they preferred their chosen option, or no action be taken. Preferences were analysed using conditional logistic regressions-based on random utility models.

**Results:** Little heterogeneity existed in the preferences of different stakeholders from different demographic groups. For the most part, reducing deer numbers in high risk CWD areas through the increased use of hunters was preferred to using government sharpshooters. Likewise, restricting the movement of carcasses and hunted products was also generally positively received by most groups, with the notable exception of hunters. There were also options that were almost universally disliked. For example, increasing incentives for private landowners to allow hunting on their lands, by allowing them to charge hunters for access, was even disliked by landowners. We also find that most respondents prefer some action to inaction.

**Conclusions:** As CWD increases in spread and prevalence, policymakers are deciding which management options should be pursued. Our results provide policymakers with important information regarding
<figure><img src="image_129.png" /></figure>

public preferences for some management options that are available for CWD. When formulating management plans, consideration of these preferences could help establish and maintain public support for addressing CWD. With changing technology regarding the monitoring of CWD, new options are emerging that will warrant further inquiries into public preferences.

**KEYWORDS:** Chronic wasting disease; preferences; paired comparison

## 226. Efficient RT-QuIC seeding activity for α-synuclein in olfactory mucosa samples of patients with Parkinson's disease and Multiple System Atrophy

Chiara Maria Giulia De Lucaª, Antonio Emanuele Eliaᵇ, Sara Maria Portaleoneᶜ, Federico Angelo Cazzanigaª, Martina Rossiᵈ, Edoardo Bistaffaª, Elena De Ceccoᵈ, Joanna Narkiewiczᵈ, Giulia Salzanoᵈ, Olga Carlettaª, Luigi Romitoᵇ, Grazia Devigiliᵇ, Paola Soliveriᵇ, Pietro Tiraboschiª, Giuseppe Legnameᵈ, Fabrizio Tagliaviniᵉ, Roberto Eleopraᵇ, Giorgio Giacconeª and Fabio Modaª

CONTACT Fabio Moda fabio.moda@istituto-besta.it

### ABSTRACT

Parkinson's disease (PD) is a neurodegenerative disorder whose diagnosis is often challenging because symptoms may overlap with neurodegenerative parkinsonisms. PD is characterized by intraneuronal accumulation of abnormal α-synuclein in brainstem while neurodegenerative parkinsonisms might be associated with accumulation of either α-synuclein, as in the case of Multiple System Atrophy (MSA) or tau, as in the case of Corticobasal Degeneration (CBD) and Progressive Supranuclear Palsy (PSP), in other disease-specific brain regions. Their definite diagnosis can be formulated only neuropathologically by detection and localization of these aggregates, considered disease-specific biomarkers (DSB), in the brain. Compelling evidence shows that trace amount of DSB can appear in peripheral tissues, including receptor neurons of the olfactory mucosa (OM), but their concentration is well below the limits of detection of the conventional diagnostic techniques.

We have therefore exploited the ultrasensitive Real Time Quaking Induced Conversion (RT-QuIC) assay for the analysis of OM collected from patients with a clinical diagnosis of PD, MSA, CBD and PSP. Our results showed that PD and MSA samples induced stronger RT-QuIC seeding activity for α-synuclein compared to CBD and PSP samples. Notably, the final RT-QuIC aggregates obtained from MSA and PD samples demonstrated peculiar biochemical and morphological features useful for their discrimination. These results suggest that RT-QuIC represents an important tool for the diagnostic workup for PD and neurodegenerative parkinsonisms

**KEYWORDS:** RT-QuIC; olfactory mucosa; α-synuclein; Parkinson's Disease; parkinsonisms

## 227. Investigating the effect of familial mutations in tau on the conformation and biological activity of tau prions

Victor Banerjeeª, Gregory E. Mertzª, Eric Tseª,ᵇ, Nick A. Parasª,ᶜ, Amanda L. Woermanª,ᶜ, Daniel R. Southworthª,ᵇ, and Stanley B. Prusinerª,ᵇ,ᶜ

Abstract not available.

## 228. Cervid Prnp polymorphism at codon 116 generates new and distinct CWD strains

Samia Hannaouiª, Elizabeth Triscottᵇ, Camilo Duque Velásquezᵇ, Debbie McKenzieᵇ, Sabine Gilchª

### ABSTRACT

Chronic wasting disease (CWD) is a prion disease of cervids. To assess the role of strain emergence and adaptation in CWD, we have characterized the impact of the 116 (A >G) polymorphism of the white-tailed deer (WTD) prion protein on CWD infection. Previously, we compared CWD prion isolates from WTD encoding wild-type (116AA) or polymorphic (116AG) PrP<sup>C</sup>. We found that 116AG-prions were conformationally less stable and more sensitive to proteases; infection of primary neuronal cultures with these isolates showed reduced infectivity

<footnote>ªUnit of Neurology 5 and Neuropathology, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milano, Italy; ᵇUnit of Neurology I – Parkinson and Movement Disorders Unit, Fondazione IRCCS Istituto Neurologico Carlo Besta, – Milano, Italy; ᶜDepartment of Health Sciences, Università degli Studi di Milano, Otolaryngology Unit, San Paolo Hospital, Milano, Italy; ᵈDepartment of Neuroscience, Scuola Internazionale Superiore di Studi Avanzati (SISSA), Laboratory of Prion Biology, Trieste, Italy; ᵉScientific Directorate, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milano, Italy.</footnote>

<footnote>ªInstitute for Neurodegenerative Diseases, Weill Institute for Neurosciences, University of California, San Francisco, San 11865 Francisco, CA, USA; ᵇDepartment of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA, USA; ᶜDepartment of Neurology, University of California, San Francisco, San Francisco, CA, USA</footnote>

<footnote>ªDepartment of Ecosystem and Public Health, Calgary Prion Research Unit, Faculty of Veterinary Medicine; Hotchkiss Brain Institute; University of Calgary, Calgary, Alberta, Canada; ᵇCenter for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Alberta, Canada</footnote>
<figure><img src="image_133.png" /></figure>

public preferences for some management options that are available for CWD. When formulating management plans, consideration of these preferences could help establish and maintain public support for addressing CWD. With changing technology regarding the monitoring of CWD, new options are emerging that will warrant further inquiries into public preferences.

**KEYWORDS:** Chronic wasting disease; preferences; paired comparison

## 226. Efficient RT-QuIC seeding activity for α-synuclein in olfactory mucosa samples of patients with Parkinson's disease and Multiple System Atrophy

Chiara Maria Giulia De Lucaª, Antonio Emanuele Eliaᵇ, Sara Maria Portaleoneᶜ, Federico Angelo Cazzanigaª, Martina Rossiᵈ, Edoardo Bistaffaª, Elena De Ceccoᵈ, Joanna Narkiewiczᵈ, Giulia Salzanoᵈ, Olga Carlettaª, Luigi Romitoᵇ, Grazia Devigiliᵇ, Paola Soliveriᵇ, Pietro Tiraboschiª, Giuseppe Legnameᵈ, Fabrizio Tagliaviniᵉ, Roberto Eleopraᵇ, Giorgio Giacconeª and Fabio Modaª

CONTACT Fabio Moda fabio.moda@istituto-besta.it

### ABSTRACT

Parkinson's disease (PD) is a neurodegenerative disorder whose diagnosis is often challenging because symptoms may overlap with neurodegenerative parkinsonisms. PD is characterized by intraneuronal accumulation of abnormal α-synuclein in brainstem while neurodegenerative parkinsonisms might be associated with accumulation of either α-synuclein, as in the case of Multiple System Atrophy (MSA) or tau, as in the case of Corticobasal Degeneration (CBD) and Progressive Supranuclear Palsy (PSP), in other disease-specific brain regions. Their definite diagnosis can be formulated only neuropathologically by detection and localization of these aggregates, considered disease-specific biomarkers (DSB), in the brain. Compelling evidence shows that trace amount of DSB can appear in peripheral tissues, including receptor neurons of the olfactory mucosa (OM), but their concentration is well below the limits of detection of the conventional diagnostic techniques.

We have therefore exploited the ultrasensitive Real Time Quaking Induced Conversion (RT-QuIC) assay for the analysis of OM collected from patients with a clinical diagnosis of PD, MSA, CBD and PSP. Our results showed that PD and MSA samples induced stronger RT-QuIC seeding activity for α-synuclein compared to CBD and PSP samples. Notably, the final RT-QuIC aggregates obtained from MSA and PD samples demonstrated peculiar biochemical and morphological features useful for their discrimination. These results suggest that RT-QuIC represents an important tool for the diagnostic workup for PD and neurodegenerative parkinsonisms

**KEYWORDS:** RT-QuIC; olfactory mucosa; α-synuclein; Parkinson's Disease; parkinsonisms

## 227. Investigating the effect of familial mutations in tau on the conformation and biological activity of tau prions

Victor Banerjeeª, Gregory E. Mertzª, Eric Tseª,ᵇ, Nick A. Parasª,ᶜ, Amanda L. Woermanª,ᶜ, Daniel R.Southworthª,ᵇ, and Stanley B. Prusinerª,ᵇ,ᶜ

Abstract not available.

## 228. Cervid Prnp polymorphism at codon 116 generates new and distinct CWD strains

Samia Hannaouiª, Elizabeth Triscottᵇ, Camilo Duque Velásquezᵇ, Debbie McKenzieᵇ, Sabine Gilchª

### ABSTRACT

Chronic wasting disease (CWD) is a prion disease of cervids. To assess the role of strain emergence and adaptation in CWD, we have characterized the impact of the 116 (A >G) polymorphism of the white-tailed deer (WTD) prion protein on CWD infection. Previously, we compared CWD prion isolates from WTD encoding wild-type (116AA) or polymorphic (116AG) PrP<sup>C</sup>. We found that 116AG-prions were conformationally less stable and more sensitive to proteases; infection of primary neuronal cultures with these isolates showed reduced infectivity

<footnote>ªUnit of Neurology 5 and Neuropathology, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milano, Italy; ᵇUnit of Neurology I – Parkinson and Movement Disorders Unit, Fondazione IRCCS Istituto Neurologico Carlo Besta, – Milano, Italy; ᶜDepartment of Health Sciences, Università degli Studi di Milano, Otolaryngology Unit, San Paolo Hospital, Milano, Italy; ᵈDepartment of Neuroscience, Scuola Internazionale Superiore di Studi Avanzati (SISSA), Laboratory of Prion Biology, Trieste, Italy; ᵉScientific Directorate, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milano, Italy.</footnote>

<footnote>ªInstitute for Neurodegenerative Diseases, Weill Institute for Neurosciences, University of California, San Francisco, San 11865 Francisco, CA, USA; ᵇDepartment of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA, USA; ᶜDepartment of Neurology, University of California, San Francisco, San Francisco, CA, USA</footnote>

<footnote>ªDepartment of Ecosystem and Public Health, Calgary Prion Research Unit, Faculty of Veterinary Medicine; Hotchkiss Brain Institute; University of Calgary, Calgary, Alberta, Canada; ᵇCenter for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Alberta, Canada</footnote>
associated with the WTD-116AG isolate and a lower seed-
ing activity in a cell-free conversion assay. Molecular
dynamics simulations suggested that the structure of
116G-PrP<sup>C</sup> was more flexible than 116A-PrP<sup>C</sup>.

To understand the transmission and strain properties of
the WTD-116AG isolate, we serially passaged it and the
WTD-116AA (Wisc-1) prions in tg(CerPrP)1536<sup>+/+</sup> mice
overexpressing deer 116AA-PrP<sup>C</sup>. Upon the first passage,
the 116AG isolate had a longer incubation period than the
Wisc-1 isolate; interestingly, the differences in biochemical
properties between Wisc-1 and WTD- 116AG were
retained supporting the characterization of at least one
new prion strain amongst the 116AG-prions.

Upon secondary passage, infectivity of 116AG-prions
was significantly enhanced in 80% of the mice and the
conformational features were retained. Remarkably, this
passage allowed us to distinguish two populations of mice
with a short and a long incubation period. Although on
first passage, only one homogenous population was
obvious, subsequent passages allowed a better distinction
and characterization of two 116AG-prions (short and long
incubation). Biological cloning in tg(CerPrP)1536<sup>+/+</sup> mice
through inoculation of 10-fold serial dilutions of the
116AA and 116AG deer brain homogenates confirmed
the presence of a second strain in the 116AG isolate, yet
the slow disease progression feature of the polymorphic
isolate was still striking for both of the 116AG-prions
(short and long incubation) compared to the rapid disease
progression of the 116AA. Interestingly, upon deglycosyla-
tion, PK-resistant fragments with different electrophoretic
mobility are observed. Further confirmation of two strains
present in 116AG was obtained following passage of this
inocula in Syrian Golden Hamsters. Ten of twelve hamsters
infected with 116AG were positive for PrP<sup>res</sup>. Western blot
analysis of PrP<sup>res</sup> showed two different migration patterns,
high molecular weight (7/10) and low molecular weight
migration pattern (3/10). Our data support the hypothesis
that two strains co-existed, as dominant and minor strains,
in the 116AG deer.

Our findings give insight into the variability of CWD
strains, which can have important implications in terms
of their possible distinct capacities to cross species
barriers into both cervid and non-cervids.

229. Aberrations of Amyloid-β in slow and rapidly progressive dementias

Aneeqa Noor<sup>a*</sup>, Saima Zafar<sup>a*</sup>, Hassan Dihazi<sup>b</sup> and Inga Zerr<sup>a</sup>

CONTACT Aneeqa Noor aneeqa.noor@med.uni-goettingen.de

ABSTRACT

Amyloid-β (Aβ), one of the key players in Alzheimer's disease (AD), has the capability to exist as various proteoforms [1]. Over the last decade the self-seeding capability and relative pathogenicity of these proteoforms has been an active target of research however, their distinct clinical involvement in neurodegeneration is yet to be elucidated. For this purpose, we aimed to decipher the prion-like structural and functional aspects of Aβ aggregation in rapidly progressive dementias.

We used affinity-based proteomic profiling to isolate
Aβ from brains of AD, rapidly progressive AD (rpAD)
and non-demented controls and established a signature of
brain-derived proteoforms, adding to the evidence that
Aβ₄₀ and Aβ₄₂ are not sole players in progression of AD.
Our preliminary experiments reveal a differential involve-
ment of 26 different proteoforms in AD and rpAD brain.
Quantification of various Aβ cleaving enzymes in the
same samples supported the generation of distinct proteo-
forms in AD and rpAD. To further understand the patho-
logical significance of Aβ in slow and rapidly progressive
dementias, we isolated interacting partners of Aβ from
AD, rpAD, non-demented controls and Creutzfeldt-Jacob
Disease (CJD) brains. In addition to commonly known
partners like Tau and Cellular Prion Protein, we were able
to isolate around 400 different proteins that showed a
disease specific involvement. Primary targets were found
to be involved in energy metabolism, neurotransmission,
neuronal development and stress response. Assessment of
the clinical relevance of these aberrations and interactions
is currently underway in various AD models.

Our study presents a novel insight in the Aβ-based
differences among slow and rapidly progressive demen-
tias, primarily the rapidly progressive variant of AD. In
addition to providing the evidence that rpAD features
the involvement of discrete molecular machinery, it
also provides insights into the players involved in pla-
cing dementias on a course of rapid progression.

Reference

[1] Portelius, et al. *Acta Neuropathol*. 2010;120:185-193.

230. Critical structural and kinetic impact of cofactors on host-to-host transmissibility of human prions

Jiri Safar, Chase Kim, Shugui Chen, Tracy Haldiman,
Vitautas Smirnovas, Krystyna Surewicz, Nicholas

<footnote><sup>a</sup>Prion Research Group, National Reference Center for TSE Surveillance, Department of Neurology, Georg-August University, Göttingen, Germany; <sup>b</sup>Department of Nephrology and Rheumatology, Georg-August University, Göttingen, Germany</footnote>

<footnote>*These authors contributed equally to this work</footnote>
associated with the WTD-116AG isolate and a lower seed-
ing activity in a cell-free conversion assay. Molecular
dynamics simulations suggested that the structure of
116G-PrP<sup>C</sup> was more flexible than 116A-PrP<sup>C</sup>.

To understand the transmission and strain properties of
the WTD-116AG isolate, we serially passaged it and the
WTD-116AA (Wisc-1) prions in tg(CerPrP)1536<sup>+/+</sup> mice
overexpressing deer 116AA-PrP<sup>C</sup>. Upon the first passage,
the 116AG isolate had a longer incubation period than the
Wisc-1 isolate; interestingly, the differences in biochemical
properties between Wisc-1 and WTD- 116AG were
retained supporting the characterization of at least one
new prion strain amongst the 116AG-prions.

Upon secondary passage, infectivity of 116AG-prions
was significantly enhanced in 80% of the mice and the
conformational features were retained. Remarkably, this
passage allowed us to distinguish two populations of mice
with a short and a long incubation period. Although on
first passage, only one homogenous population was
obvious, subsequent passages allowed a better distinction
and characterization of two 116AG-prions (short and long
incubation). Biological cloning in tg(CerPrP)1536<sup>+/+</sup> mice
through inoculation of 10-fold serial dilutions of the
116AA and 116AG deer brain homogenates confirmed
the presence of a second strain in the 116AG isolate, yet
the slow disease progression feature of the polymorphic
isolate was still striking for both of the 116AG-prions
(short and long incubation) compared to the rapid disease
progression of the 116AA. Interestingly, upon deglycosyla-
tion, PK-resistant fragments with different electrophoretic
mobility are observed. Further confirmation of two strains
present in 116AG was obtained following passage of this
inocula in Syrian Golden Hamsters. Ten of twelve hamsters
infected with 116AG were positive for PrP<sup>res</sup>. Western blot
analysis of PrP<sup>res</sup> showed two different migration patterns,
high molecular weight (7/10) and low molecular weight
migration pattern (3/10). Our data support the hypothesis
that two strains co-existed, as dominant and minor strains,
in the 116AG deer.

Our findings give insight into the variability of CWD
strains, which can have important implications in terms
of their possible distinct capacities to cross species
barriers into both cervid and non-cervids.

229. Aberrations of Amyloid-β in slow and rapidly progressive dementias

Aneeqa Noor<sup>a*</sup>, Saima Zafar<sup>a*</sup>, Hassan Dihazi<sup>b</sup> and Inga Zerr<sup>a</sup>

CONTACT Aneeqa Noor aneeqa.noor@med.uni-goettingen.de

ABSTRACT

Amyloid-β (Aβ), one of the key players in Alzheimer's disease (AD), has the capability to exist as various proteoforms [1]. Over the last decade the self-seeding capability and relative pathogenicity of these proteoforms has been an active target of research however, their distinct clinical involvement in neurodegeneration is yet to be elucidated. For this purpose, we aimed to decipher the prion-like structural and functional aspects of Aβ aggregation in rapidly progressive dementias.

We used affinity-based proteomic profiling to isolate
Aβ from brains of AD, rapidly progressive AD (rpAD)
and non-demented controls and established a signature of
brain-derived proteoforms, adding to the evidence that
Aβ₄₀ and Aβ₄₂ are not sole players in progression of AD.
Our preliminary experiments reveal a differential involve-
ment of 26 different proteoforms in AD and rpAD brain.
Quantification of various Aβ cleaving enzymes in the
same samples supported the generation of distinct proteo-
forms in AD and rpAD. To further understand the patho-
logical significance of Aβ in slow and rapidly progressive
dementias, we isolated interacting partners of Aβ from
AD, rpAD, non-demented controls and Creutzfeldt-Jacob
Disease (CJD) brains. In addition to commonly known
partners like Tau and Cellular Prion Protein, we were able
to isolate around 400 different proteins that showed a
disease specific involvement. Primary targets were found
to be involved in energy metabolism, neurotransmission,
neuronal development and stress response. Assessment of
the clinical relevance of these aberrations and interactions
is currently underway in various AD models.

Our study presents a novel insight in the Aβ-based
differences among slow and rapidly progressive demen-
tias, primarily the rapidly progressive variant of AD. In
addition to providing the evidence that rpAD features
the involvement of discrete molecular machinery, it
also provides insights into the players involved in pla-
cing dementias on a course of rapid progression.

Reference

[1] Portelius, et al. *Acta Neuropathol*. 2010;120:185-193.

230. Critical structural and kinetic impact of cofactors on host-to-host transmissibility of human prions

Jiri Safar, Chase Kim, Shugui Chen, Tracy Haldiman,
Vitautas Smirnovas, Krystyna Surewicz, Nicholas

<footnote><sup>a</sup>Prion Research Group, National Reference Center for TSE Surveillance, Department of Neurology, Georg-August University, Göttingen, Germany; <sup>b</sup>Department of Nephrology and Rheumatology, Georg-August University, Göttingen, Germany</footnote>

<footnote>*These authors contributed equally to this work</footnote>
<figure><img src="image_130.png" /></figure>

Maurer, Qingzhong Kong, Qinzhong Kong and Witold Surewicz

Case Western, Reserve University

## ABSTRACT

**Objectives:** Molecular characteristics determining the serial host-to-host transmissibility of human prions causing Creutzfeldt-Jakob disease (CJD) are unknown, and several recent clinical therapeutic trials failed. The key reasons behind these disappointing results are (a) fundamental structural differences between human prions causing diseases and cloned laboratory prions, and (b) distinct replication mechanism of human prions that is not determined by their conformational stability, as it is in yeast and some murine prions, but by growth rate of prion aggregates, which is in turn controlled by their specific structural features. How these aspects impact transmissibility of prions to man is an imperative question in light of history of iatrogenic and zoonotic transmissions and recent findings of human prions in the skin and body fluids.

**Methods:** We investigated the effect of distinct cofactors on the structure and replication kinetics of major human prion strains. Using these data, we synthesized with variable cofactors a series of new human prions from the recombinant human prion protein, and to assess the impact of glycosylation, monitored their replication rate and infectivity in serial passage in transgenic mice that were expressing physiological levels of human prion proteins with or without glycosylation.

**Results:** We report that different lipid and polyanionic cofactors have a major structural and kinetic impact on the human prion replication *in vitro*, infectivity *in vivo*, and strain characteristics. Within the synthetic prion spectrum, recombinant human prion generated with ganglioside GM1 cofactor caused unique and stable neurologic dysfunction with incubation times comparable to natural CJD prion strain in both first and second passage, respectively. Unique hybrid MM1/MM2 CJD-like prion neuropathology, high replication rate of reisolated prions, and biophysical profiling indicate that a novel particularly infectious and aggressive human prion strain was created.

**Conclusions:** Lipid and polyanion cofactors have fundamental impact on human prion strain characteristics and transmissibility. Their seemingly subtle effects on structural organization of prions are coupled with large differences in replication rates, and together with posttranslational modifications, play a crucial role as determinants of infectivity, host range, and extraordinary precision in targeting specific brain structures.

# 231. Prp1 is necessary for seizure-susceptibility in appa zebrafish knockout and new in vivo anti-convulsants

Richard Kanyoª, Laszlo Locskaiª, Patricia L. A. Leightonª, Harley T. Kuratab and W. Ted Allisonª, c

CONTACT Richard Kanyo rkanyo@ualberta.ca

## ABSTRACT

**Background:** Cellular prion protein (PrPC) has been linked to Alzheimer's disease (AD), the most prevalent dementia. PrPC directly interacts with amyloid-β oligomers and amyloid precursor protein (APP) and the genetic interaction plus mechanism was further explored here. Because 10-22% of AD patients display epileptic episodes, potential anti-convulsants, ML213 and ICA73, which target Kv7.2/3 channels have been investigated *in vivo*. Zebrafish enable economical high-throughput drug screening platforms that faithfully represent the complexity of the central nervous system and are therefore an excellent model for these goals.

**Methods:** appa;prp1 compound mutant (*Appa* and *Prp1* are disrupted) fish were engineered by targeted mutagenesis. Seizure-susceptibility was compared in mutant and wild type larvae via treatment with convulsants, pentylenetetrazol (PTZ) or 4-aminopyridine (4AP). Locomotor activity was tracked using Ethovision behavioural tracking software. Neural activity of free-swimming animals was measured using the novel ratiometric fluorescence-based Ca2+ indicator CaMPARI.

**Results:** PTZ dose-responses of locomotor activity revealed that *Appa* and *Prp1* single mutants exhibit enhanced seizure-susceptibility compared to wild type fish. However, seizure-susceptibility is dampened/ reduced in compound mutants compared to either individual mutant. Measuring neural activity with CaMPARI, and using 4AP as a convulsant, ML213 and ICA73 were demonstrated to have anti-convulsant activity *in vivo*. ML213 was a very potent anti-convulsant, whereas ICA73 had similar potency to retigabine (RTG), an FDA approved drug used previously to treat epilepsy. When measuring locomotor activity, ML213 and RTG paralleled our results obtained with CaMPARI, but ICA73 did not and displayed an increase in locomotor activity highlighting the importance of using multiple assays to complement one another.

<footnote>ªCentre for Prions & Protein Folding Disease, and Department of Biological Sciences, University of Alberta, Edmonton, AB, Canada;</footnote>

<footnote>ᵇDepartment of Pharmacology, University of Alberta, Edmonton, AB, Canada;</footnote>

<footnote>ᶜDepartment of Medical Genetics, University of Alberta, Edmonton AB, CANADA</footnote>
<figure><img src="image_134.png" /></figure>

Maurer, Qingzhong Kong, Qinzhong Kong and Witold Surewicz

Case Western, Reserve University

## ABSTRACT

**Objectives:** Molecular characteristics determining the serial host-to-host transmissibility of human prions causing Creutzfeldt-Jakob disease (CJD) are unknown, and several recent clinical therapeutic trials failed. The key reasons behind these disappointing results are (a) fundamental structural differences between human prions causing diseases and cloned laboratory prions, and (b) distinct replication mechanism of human prions that is not determined by their conformational stability, as it is in yeast and some murine prions, but by growth rate of prion aggregates, which is in turn controlled by their specific structural features. How these aspects impact transmissibility of prions to man is an imperative question in light of history of iatrogenic and zoonotic transmissions and recent findings of human prions in the skin and body fluids.

**Methods:** We investigated the effect of distinct cofactors on the structure and replication kinetics of major human prion strains. Using these data, we synthesized with variable cofactors a series of new human prions from the recombinant human prion protein, and to assess the impact of glycosylation, monitored their replication rate and infectivity in serial passage in transgenic mice that were expressing physiological levels of human prion proteins with or without glycosylation.

**Results:** We report that different lipid and polyanionic cofactors have a major structural and kinetic impact on the human prion replication *in vitro*, infectivity *in vivo*, and strain characteristics. Within the synthetic prion spectrum, recombinant human prion generated with ganglioside GM1 cofactor caused unique and stable neurologic dysfunction with incubation times comparable to natural CJD prion strain in both first and second passage, respectively. Unique hybrid MM1/MM2 CJD-like prion neuropathology, high replication rate of reisolated prions, and biophysical profiling indicate that a novel particularly infectious and aggressive human prion strain was created.

**Conclusions:** Lipid and polyanion cofactors have fundamental impact on human prion strain characteristics and transmissibility. Their seemingly subtle effects on structural organization of prions are coupled with large differences in replication rates, and together with posttranslational modifications, play a crucial role as determinants of infectivity, host range, and extraordinary precision in targeting specific brain structures.

# 231. Prp1 is necessary for seizure-susceptibility in appa zebrafish knockout and new in vivo anti-convulsants

Richard Kanyoª, Laszlo Locskaiª, Patricia L. A. Leightonª, Harley T. Kuratab and W. Ted Allisonª, c

CONTACT Richard Kanyo rkanyo@ualberta.ca

## ABSTRACT

**Background:** Cellular prion protein (PrPC) has been linked to Alzheimer's disease (AD), the most prevalent dementia. PrPC directly interacts with amyloid-β oligomers and amyloid precursor protein (APP) and the genetic interaction plus mechanism was further explored here. Because 10-22% of AD patients display epileptic episodes, potential anti-convulsants, ML213 and ICA73, which target Kv7.2/3 channels have been investigated *in vivo*. Zebrafish enable economical high-throughput drug screening platforms that faithfully represent the complexity of the central nervous system and are therefore an excellent model for these goals.

**Methods:** appa; prp1 compound mutant (*Appa* and *Prp1* are disrupted) fish were engineered by targeted mutagenesis. Seizure-susceptibility was compared in mutant and wild type larvae via treatment with convulsants, pentylenetetrazol (PTZ) or 4-aminopyridine (4AP). Locomotor activity was tracked using Ethovision behavioural tracking software. Neural activity of free-swimming animals was measured using the novel ratiometric fluorescence-based Ca2+ indicator CaMPARI.

**Results:** PTZ dose-responses of locomotor activity revealed that *Appa* and *Prp1* single mutants exhibit enhanced seizure-susceptibility compared to wild type fish. However, seizure-susceptibility is dampened/ reduced in compound mutants compared to either individual mutant. Measuring neural activity with CaMPARI, and using 4AP as a convulsant, ML213 and ICA73 were demonstrated to have anti-convulsant activity *in vivo*. ML213 was a very potent anti-convulsant, whereas ICA73 had similar potency to retigabine (RTG), an FDA approved drug used previously to treat epilepsy. When measuring locomotor activity, ML213 and RTG paralleled our results obtained with CaMPARI, but ICA73 did not and displayed an increase in locomotor activity highlighting the importance of using multiple assays to complement one another.

<footnote>ªCentre for Prions & Protein Folding Disease, and Department of Biological Sciences, University of Alberta, Edmonton, AB, Canada;</footnote>

<footnote>ᵇDepartment of Pharmacology, University of Alberta, Edmonton, AB, Canada;</footnote>

<footnote>ᶜDepartment of Medical Genetics, University of Alberta, Edmonton AB, CANADA</footnote>
<figure><img src="image_131.png" /></figure>

**Conclusion:** *appa* and *prp1* mutants both exhibited enhanced seizure-susceptibility. Interestingly, *prp1* function was required for maximum seizure-susceptibility in *appa* mutants suggesting a mechanism/pathway by which *prp1* functions as a rheostat of seizure-susceptibility or neural hyperactivity. Therefore a bi-functional mechanism may explain our recently published findings [1], where compound *prp1; prp2* mutants are more susceptible to seizures than *prp1* mutants, but less than *prp2* mutants. ML213 and ICA73 were established here as anti-convulsants *in vivo* and thus may provide a route in future mechanistic studies to gain sufficient insight and treat AD patients that display seizures. Together, this work using engineered zebrafish and anti-convulsants could illuminate the PrP-APP pathway as a potential aetiological link between AD and epilepsy.

## Reference

[1] Leighton, Kanyo et al. JBC. 2018;293:12576-12592.

## 232. 'Novel compounds for the treatment of prion disease'

Robert C.C. Mercerª, Nhat T. T. Leª, Doug Fraserᵇ, Aaron Beelerᵇ and David A. Harrisª

ªBoston University School of Medicine, Department of Biochemistry; ᵇBoston University, Department of Chemistry

## ABSTRACT

Prion diseases are invariably fatal neurodegenerative diseases of humans and other animals for which there are no currently available treatments. Using a prion infected mouse neuroblastoma cell line (ScN2a) as an assay system, many anti-prion compounds have been identified, but none have resulted in a clinically effective therapy. Interestingly, while several anti-prion compounds exert their effects through a direct interaction with PrP<sup>C</sup> or PrP<sup>Sc</sup>, the mechanisms underlying the anti-prion effects of other compounds remains enigmatic. A recent report from our laboratory [1] describes the identification of a novel class of anti-prion compounds, phenethyl piperidines. These were discovered using a high throughput screen for molecules that prevent mutant PrP toxicity. JZ107, the most potent derivative, can permanently cure ScN2a cells of infection and rescue hippocampal neurons from PrP<sup>Sc</sup> induced synaptotoxicity *in vitro*. Further study of JZ107 has led, unexpectedly, to the discovery of a number of additional, novel anti-prion compounds, some of which have previously identified molecular targets and have been used in other clinical

settings. None of these compounds bind directly to PrP, making the identity of their interaction partners of significant interest. To further validate the anti-prion effects of these compounds, we assessed their ability to prevent the retraction of hippocampal neuron dendritic spines following exposure to purified prion preparations. We have begun assessing the anti-prion effects of the most potent of these compounds *in vivo*.

## Reference

[1] Imberdis T, et al. Identification of anti-prion compounds using a novel cellular assay. JBC. 2016;291:26164-26176.

## 233. The many phases of prion protein as observed by NMR

Lauren E. Kleinª, Mikail A. Kostelovᵇ, Marcus D. Tuttleª, Stephen M. Strittmatterᵇ and Kurt W. Zilmª

ªDepartment of Chemistry, Yale University, New Haven, Connecticut, USA; ᵇDepartment of Neurology, Yale School of Medicine, New Haven, Connecticut, USA

CONTACT Kurt W. Zilm kurt.zilm@yale.edu

## ABSTRACT

Nuclear magnetic resonance (NMR) has proven invaluable in characterizing all sorts of functionally important macromolecular aggregates from amyloids to membrane-less organelles. In this contribution we describe the discovery and characterization by NMR of multiple phase states of prion protein [1] with relevance to Alzheimer's disease (AD). The binding of amyloid-beta oligomers (A β o) to PrP<sup>C</sup> has been demonstrated to lead to synapse loss and the onset of symptoms in an animal model of AD. Biophysical investigation of the mode of PrP<sup>C</sup>/A β o association found formation of a stoichiometric complex. When studied by magic angle spinning (MAS) NMR, this condensate was revealed to be a hydrogel phase. We find liquid-like mobility for PrP<sup>C</sup> in the hydrogel, while the A β o instead behave as a relatively rigid molecular solid. Association of PrP<sup>C</sup> to A β o is mediated via lysine clusters, which can be detected by observation of large differential mobility between bound and unbound lysines by <sup>15</sup>N MAS NMR. Exploration of the PrP<sup>C</sup>/ A β o phase space further lead to discovery of a PrP<sup>C</sup> rich viscous liquid phase, also studied by <sup>13</sup>C MAS NMR methods.

Repetitive motifs and clustering of identical residues makes PrP<sup>C</sup> a difficult target for NMR resonance assignment. Nevertheless, this same attribute makes it
<figure><img src="image_135.png" /></figure>

**Conclusion:** *appa* and *prp1* mutants both exhibited enhanced seizure-susceptibility. Interestingly, *prp1* function was required for maximum seizure-susceptibility in *appa* mutants suggesting a mechanism/pathway by which *prp1* functions as a rheostat of seizure-susceptibility or neural hyperactivity. Therefore a bi-functional mechanism may explain our recently published findings [1], where compound *prp1;prp2* mutants are more susceptible to seizures than *prp1* mutants, but less than *prp2* mutants. ML213 and ICA73 were established here as anti-convulsants *in vivo* and thus may provide a route in future mechanistic studies to gain sufficient insight and treat AD patients that display seizures. Together, this work using engineered zebrafish and anti-convulsants could illuminate the PrP-APP pathway as a potential aetiological link between AD and epilepsy.

## Reference

[1] Leighton, Kanyo et al. JBC. 2018;293:12576-12592.

## 232. 'Novel compounds for the treatment of prion disease'

Robert C.C. Mercerª, Nhat T. T. Leª, Doug Fraserᵇ, Aaron Beelerᵇ and David A. Harrisª

ªBoston University School of Medicine, Department of Biochemistry; ᵇBoston University, Department of Chemistry

## ABSTRACT

Prion diseases are invariably fatal neurodegenerative diseases of humans and other animals for which there are no currently available treatments. Using a prion infected mouse neuroblastoma cell line (ScN2a) as an assay system, many anti-prion compounds have been identified, but none have resulted in a clinically effective therapy. Interestingly, while several anti-prion compounds exert their effects through a direct interaction with PrP<sup>C</sup> or PrP<sup>Sc</sup>, the mechanisms underlying the anti-prion effects of other compounds remains enigmatic. A recent report from our laboratory [1] describes the identification of a novel class of anti-prion compounds, phenethyl piperidines. These were discovered using a high throughput screen for molecules that prevent mutant PrP toxicity. JZ107, the most potent derivative, can permanently cure ScN2a cells of infection and rescue hippocampal neurons from PrP<sup>Sc</sup> induced synaptotoxicity *in vitro*. Further study of JZ107 has led, unexpectedly, to the discovery of a number of additional, novel anti-prion compounds, some of which have previously identified molecular targets and have been used in other clinical

settings. None of these compounds bind directly to PrP, making the identity of their interaction partners of significant interest. To further validate the anti-prion effects of these compounds, we assessed their ability to prevent the retraction of hippocampal neuron dendritic spines following exposure to purified prion preparations. We have begun assessing the anti-prion effects of the most potent of these compounds *in vivo*.

## Reference

[1] Imberdis T, et al. Identification of anti-prion compounds using a novel cellular assay. JBC. 2016;291:26164-26176.

## 233. The many phases of prion protein as observed by NMR

Lauren E. Kleinª, Mikail A. Kostelovᵇ, Marcus D. Tuttleª, Stephen M. Strittmatterᵇ and Kurt W. Zilmª

ªDepartment of Chemistry, Yale University, New Haven, Connecticut, USA; ᵇDepartment of Neurology, Yale School of Medicine, New Haven, Connecticut, USA

CONTACT Kurt W. Zilm kurt.zilm@yale.edu

## ABSTRACT

Nuclear magnetic resonance (NMR) has proven invaluable in characterizing all sorts of functionally important macromolecular aggregates from amyloids to membrane-less organelles. In this contribution we describe the discovery and characterization by NMR of multiple phase states of prion protein [1] with relevance to Alzheimer's disease (AD). The binding of amyloid-beta oligomers (A β o) to PrP<sup>C</sup> has been demonstrated to lead to synapse loss and the onset of symptoms in an animal model of AD. Biophysical investigation of the mode of PrP<sup>C</sup>/A β o association found formation of a stoichiometric complex. When studied by magic angle spinning (MAS) NMR, this condensate was revealed to be a hydrogel phase. We find liquid-like mobility for PrP<sup>C</sup> in the hydrogel, while the A β o instead behave as a relatively rigid molecular solid. Association of PrP<sup>C</sup> to A β o is mediated via lysine clusters, which can be detected by observation of large differential mobility between bound and unbound lysines by <sup>15</sup>N MAS NMR. Exploration of the PrP<sup>C</sup>/ A β o phase space further lead to discovery of a PrP<sup>C</sup> rich viscous liquid phase, also studied by <sup>13</sup>C MAS NMR methods.

Repetitive motifs and clustering of identical residues makes PrP<sup>C</sup> a difficult target for NMR resonance assignment. Nevertheless, this same attribute makes it
<figure><img src="image_132.png" /></figure>

possible to directly associate many resonances with
specific domains. Of the 210 aa in PrP<sup>C</sup>, 43 are glycines,
with all but 3 located in the N-terminal 21-131 seg-
ment. Six of the 8 alanines are located in the palindro-
mic AGAAAAGA run from 113-120, and 8 of the 12
threonines are located at the end of the junction
between the C-terminal helices encompassing aa 183-
201. Large scale conformational transitions can be
detected by NMR when such aa clusters exhibit simul-
taneous shifts characteristic of a change in secondary
structure. In this manner we observe both the N-term-
inal fragment and the palindromic sequence to transi-
tion from random coil to a largely helical conformation
upon A β o binding. Interestingly, the formation of the
PrP<sup>C</sup> liquid phase is found to be accompanied by a
melting of the threonine rich helical segment, and the
apparent adoption of an unusual χ<sub>1</sub> for the tryptophan
residues in the octa-repeat segment.

Biophysical characterization of these phase states of
PrP<sup>C</sup> is proving relevant to development of possible treat-
ments for AD. Compounds that disrupt the hydrogel phase
have been shown to relieve memory deficits in AD mice,
and make it possible to detect the selective dissolution of
the hydrogel phase in Alzheimer's brain lysates [2].

References

[1] Kostylev MA, et al. Mol Cell. 2018;72(3):426-443.

[2] Gunther EC, et al. Cell Rep. 2019;26(1):145-158.

234. The European Union Summary Report On Surveillance For The Presence Of Transmissible Spongiform Encephalopathies (TSE): The Situation In 2017

Angel Ortiz Pelaezª, Valentina Rizziª, Giuseppe Ruᵇ,
Francesco Ingravalleᵇ and Yves Van der Stedeª

CONTACT Angel Ortiz Pelaez angel.ortizpelaez@efsa.europa.eu

ABSTRACT

The European Food Safety Authority publishes a yearly
summary of the surveillance activities on transmissible
spongiform encephalopathies (TSE) in bovine animals,
sheep, goats, cervids, and other species, in the European
Union (EU), and in Iceland, Norway and Switzerland.
Target groups include: animals clinically suspected of
being infected by TSE; animals culled under TSE

eradication measures; animals with clinical signs at ante-
mortem; emergency slaughtered; fallen stock/not slaugh-
tered for human consumption and healthy slaughtered
animals for human consumption, for cervids also hunted
and road or predator-injured or killed.

For the first time since bovine spongiform encephalo-
pathy (BSE) had been reported, no cases of classical
BSE were reported world-wide in 2017 [1]. Six atypical
BSE cases were reported by Spain (1 H /2 L), France
(1 H/1 L) and Ireland (1 L), out of the 1,312,714 cattle
tested by 28 EU Member States (MS) and 18,526 tested
by three non-MS.

In total 431,815 small ruminants were tested in 2017 in the EU. Compared with 2016, there was a 36.2% increase in the number of cases of classical scrapie (CS) in sheep (933), mostly reported by Greece, Spain, Italy and Romania, although over 75% of the cases were sourced in infected flocks. Atypical scrapie (AS) was confirmed in 94 animals. In goats, a decrease of 10% in the number of cases of classical scrapie (567) were reported, 84% in Cyprus. Atypical scrapie was confirmed in nine animals.

Ten-year trend analysis showed a statistically significant
decrease in the sheep proportion of CS cases per 10,000
tested animals and an increase in goats. For AS, 10-year
data did not detect any statistically significant trend in both
species.

After the discovery of chronic wasting disease
(CWD) in Norway in 2016, TSE testing in cervids
increased in the EU: 10 MS tested 3,585 cervids (75%
in Romania, 98.5% from wildlife), all negative.
Norway tested 25,736 deer in 2017, leading to the
detection of the first case of CWD in a red deer, nine
cases in wild reindeer and one in a wild moose.
Following EFSA recommendations, the European
Commission introduced a 3-year mandatory surveil-
lance programme for six member states starting on 1
January 2018.

By the time of submitting this abstract, CS/AS cases
were not yet available, but one new classical BSE case
was confirmed in Scotland (2018), one L-type BSE case
in Poland (2019) and one case of CWD in a wild moose
in Finland in March 2018.

KEYWORDS: BSE; scrapie; CWD; EU; surveillance

Reference

[1] EFSA (European Food Safety Authority), 2018. The European Union summary report on surveillance for the presence of transmissible spongiform encephalopathies (TSE) in 2017. EFSA J. 2018;16(11):5492, 64 pp. DOI:10.2903/j.efsa.2018.5492

<footnote>ªUnit on Biological Hazards and Contaminants, Department of Risk
Assessment & Scientific Assistance, European Food Safety Authority
(EFSA); ᵇBiostatistics, Epidemiology and Risk Analysis (BEAR) Unit.
Istituto Zooprofilattico Sperimentale di Piemonte, Liguria e Valle d
'Aosta, Torino (Italia)</footnote>
<figure><img src="image_136.png" /></figure>

possible to directly associate many resonances with
specific domains. Of the 210 aa in PrP<sup>C</sup>, 43 are glycines,
with all but 3 located in the N-terminal 21-131 seg-
ment. Six of the 8 alanines are located in the palindro-
mic AGAAAAGA run from 113-120, and 8 of the 12
threonines are located at the end of the junction
between the C-terminal helices encompassing aa 183-
201. Large scale conformational transitions can be
detected by NMR when such aa clusters exhibit simul-
taneous shifts characteristic of a change in secondary
structure. In this manner we observe both the N-term-
inal fragment and the palindromic sequence to transi-
tion from random coil to a largely helical conformation
upon A β o binding. Interestingly, the formation of the
PrP<sup>C</sup> liquid phase is found to be accompanied by a
melting of the threonine rich helical segment, and the
apparent adoption of an unusual χ<sub>1</sub> for the tryptophan
residues in the octa-repeat segment.

Biophysical characterization of these phase states of
PrP<sup>C</sup> is proving relevant to development of possible treat-
ments for AD. Compounds that disrupt the hydrogel phase
have been shown to relieve memory deficits in AD mice,
and make it possible to detect the selective dissolution of
the hydrogel phase in Alzheimer's brain lysates [2].

References

[1] Kostylev MA, et al. Mol Cell. 2018;72(3):426-443.

[2] Gunther EC, et al. Cell Rep. 2019;26(1):145-158.

234. The European Union Summary Report On Surveillance For The Presence Of Transmissible Spongiform Encephalopathies (TSE): The Situation In 2017

Angel Ortiz Pelaezª, Valentina Rizziª, Giuseppe Ruᵇ, Francesco Ingravalleᵇ and Yves Van der Stedeª

CONTACT Angel Ortiz Pelaez angel.ortizpelaez@efsa.europa.eu

ABSTRACT

The European Food Safety Authority publishes a yearly
summary of the surveillance activities on transmissible
spongiform encephalopathies (TSE) in bovine animals,
sheep, goats, cervids, and other species, in the European
Union (EU), and in Iceland, Norway and Switzerland.
Target groups include: animals clinically suspected of
being infected by TSE; animals culled under TSE

eradication measures; animals with clinical signs at ante-
mortem; emergency slaughtered; fallen stock/not slaugh-
tered for human consumption and healthy slaughtered
animals for human consumption, for cervids also hunted
and road or predator-injured or killed.

For the first time since bovine spongiform encephalo-
pathy (BSE) had been reported, no cases of classical
BSE were reported world-wide in 2017 [1]. Six atypical
BSE cases were reported by Spain (1 H /2 L), France
(1 H/1 L) and Ireland (1 L), out of the 1,312,714 cattle
tested by 28 EU Member States (MS) and 18,526 tested
by three non-MS.

In total 431,815 small ruminants were tested in 2017 in the EU. Compared with 2016, there was a 36.2% increase in the number of cases of classical scrapie (CS) in sheep (933), mostly reported by Greece, Spain, Italy and Romania, although over 75% of the cases were sourced in infected flocks. Atypical scrapie (AS) was confirmed in 94 animals. In goats, a decrease of 10% in the number of cases of classical scrapie (567) were reported, 84% in Cyprus. Atypical scrapie was confirmed in nine animals.

Ten-year trend analysis showed a statistically significant
decrease in the sheep proportion of CS cases per 10,000
tested animals and an increase in goats. For AS, 10-year
data did not detect any statistically significant trend in both
species.

After the discovery of chronic wasting disease
(CWD) in Norway in 2016, TSE testing in cervids
increased in the EU: 10 MS tested 3,585 cervids (75%
in Romania, 98.5% from wildlife), all negative.
Norway tested 25,736 deer in 2017, leading to the
detection of the first case of CWD in a red deer, nine
cases in wild reindeer and one in a wild moose.
Following EFSA recommendations, the European
Commission introduced a 3-year mandatory surveil-
lance programme for six member states starting on 1
January 2018.

By the time of submitting this abstract, CS/AS cases
were not yet available, but one new classical BSE case
was confirmed in Scotland (2018), one L-type BSE case
in Poland (2019) and one case of CWD in a wild moose
in Finland in March 2018.

KEYWORDS: BSE; scrapie; CWD; EU; surveillance

Reference

[1] EFSA (European Food Safety Authority), 2018. The European Union summary report on surveillance for the presence of transmissible spongiform encephalopathies (TSE) in 2017. EFSA J. 2018;16(11):5492, 64 pp. DOI:10.2903/j.efsa.2018.5492

<footnote>ªUnit on Biological Hazards and Contaminants, Department of Risk
Assessment & Scientific Assistance, European Food Safety Authority
(EFSA); ᵇBiostatistics, Epidemiology and Risk Analysis (BEAR) Unit.
Istituto Zooprofilattico Sperimentale di Piemonte, Liguria e Valle d
'Aosta, Torino (Italia)</footnote>
**235. Mutations in the functional amyloid PMEL lead to clinical glaucomatous neurodegeneration: Potential for insights into avoiding amyloidosis**

W. Ted Allison<sup>a,b,c</sup>, Tim Footz<sup>b</sup>, Gavin J Neil<sup>a,c</sup> and Michael A. Walter<sup>b</sup>

<sup>a</sup>Centre for Prions & Protein Folding Disease, U Alberta, Edmonton, AB, Canada; <sup>b</sup>Department of Medical Genetics, U Alberta, Edmonton, AB, Canada; <sup>c</sup>Department of Biological Sciences, U Alberta, Edmonton, AB, Canada

CONTACT W. Ted Allison ted.allison@ualberta.ca

**ABSTRACT**

Pigmentary glaucoma (PG) is a major form of glaucoma, a leading cause of blindness worldwide. Glaucoma is neurodegeneration of retinal ganglion cells, which are neurons of the CNS. We have, for the first time, discovered a causative gene for this CNS neurodegeneration in humans. Our results reveal that mutations of the premelanosome gene (PMEL) cause PG. We identified PMEL mutations in an Alberta family with PG and in four isolated patients. In parallel, our colleagues Drs. Wiggs (Harvard Medical School) and Craig (U Flinders, Australia), identified PMEL mutations in separate PG cohorts, strongly supporting that PMEL mutations cause PG (Lahola-Chomiak et al. 2018 *Human Molecular Genetics* doi: 10.1093/hmg/ddy429) [1].

Intriguingly, PMEL protein is a rarity in vertebrates: a **functional** amyloid. PMEL forms amyloid fibril structures in pigmented cells of vertebrates, including humans, and these amyloid fibrils are structurally critical for deposition of melanin pigment. The patient PMEL mutations we identified result in impaired processing of PMEL protein and altered amyloid structure, however the central question of how these changes lead to CNS neurodegeneration remains unknown. Our zebrafish modelling of PMEL loss via CRISPR/Cas9 mutation produced recessive inheritance of swollen eye morphology in larvae that is strikingly reminiscent of increased ocular pressure in glaucoma; this implies that loss of the functional PMEL amyloid can contribute to some aspects of PG aetiology. This is in contrast with our patient pedigrees and mutations of PMEL homologs in other animals (dogs, horses, etc.) where dominant inheritance implies a toxic gain-of-function; this predicts that abnormal PMEL amyloid might lead to neurodegeneration via a (prion-like?) templating of aberrant amyloid structure.

Future work is warranted to delineate these contributions of gained toxicity vs. reduced protein function during (prion-like?) neurodegeneration [2], including defining whether both of these contributions are

required for death of CNS neurons. Such work has potential to give unique insights into amyloidosis: We identify several mutations in patients with glaucomatous neurodegeneration, and these now guide our predictions of which aspects of PMEL are the key to human CNS cells thriving through a lifetime of high amyloid content.

**References**

[1] Lahola-Chomiak, et al. Hum Mol Genet. 2018. DOI:10.1093/hmg/ddy429

[2] Allison, et al. Int J Mol Sci. 2017;18:E2223. DOI:10.3390/ijms18102223

SCHMITT-ULMS, Gerold 2019

**236. A mechanism-based small-molecule approach to the treatment of prion diseases that targets $PrP^C$**

M Mehrabian<sup>a,b</sup>, D Williams<sup>a,b</sup>, X Wang<sup>a,b,c</sup>, F Ghodrati<sup>a,b,c</sup>, M Güneş,<sup>a,b</sup> and G Schmitt-Ulms<sup>a,b,c</sup>

<sup>a</sup>Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Ontario, Canada; <sup>b</sup>Krembil Discovery Centre, Toronto, Ontario, Canada; <sup>c</sup>Department of Laboratory Medicine & Pathobiology, University of Toronto, Toronto, Ontario, Canada

**ABSTRACT**

A wide range of observations in humans and animals indicate that a reduction in steady-state levels of the cellular prion protein ($PrP^C$) is both safe and would delay or prevent prion diseases. To derive rational approaches for suppressing $PrP^C$ levels at the plasma membrane, we and others have been studying the molecular environment and function of $PrP^C$.

This work led us to recognize that prion genes evolved from a ZIP zinc transporter ancestral molecule with roles in a morphogenetic programme known as epithelial-to-mesenchymal transition (EMT). The latter guided us to investigate the possible involvement of $PrP^C$ in EMT, revealing $PrP^C$ to control the polysialylation of the neural cell adhesion molecule 1 (NCAM1), a well-studied post-translational modification that serves as a molecular marker of EMT. In several cell models we interrogated, the molecular environment of $PrP^C$ is characterized by its residence within a specialized membrane domain that hosts, in addition to NCAM1, distinct subsets of more than 20 additional proteins. Interestingly, most of these proteins have known roles in the modulation of TGFB1 and integrin sister signalling complexes that are activated during EMT.

The binding of ligands to their cognate receptors often induces the internalization of ligand-receptor
**235. Mutations in the functional amyloid PMEL lead to clinical glaucomatous neurodegeneration: Potential for insights into avoiding amyloidosis**

W. Ted Allison<sup>a,b,c</sup>, Tim Footz<sup>b</sup>, Gavin J Neil<sup>a,c</sup> and Michael A. Walter<sup>b</sup>

<sup>a</sup>Centre for Prions & Protein Folding Disease, U Alberta, Edmonton, AB, Canada; <sup>b</sup>Department of Medical Genetics, U Alberta, Edmonton, AB, Canada; <sup>c</sup>Department of Biological Sciences, U Alberta, Edmonton, AB, Canada

CONTACT W. Ted Allison ted.allison@ualberta.ca

**ABSTRACT**

Pigmentary glaucoma (PG) is a major form of glaucoma, a leading cause of blindness worldwide. Glaucoma is neurodegeneration of retinal ganglion cells, which are neurons of the CNS. We have, for the first time, discovered a causative gene for this CNS neurodegeneration in humans. Our results reveal that mutations of the premelanosome gene (PMEL) cause PG. We identified PMEL mutations in an Alberta family with PG and in four isolated patients. In parallel, our colleagues Drs. Wiggs (Harvard Medical School) and Craig (U Flinders, Australia), identified PMEL mutations in separate PG cohorts, strongly supporting that PMEL mutations cause PG (Lahola-Chomiak et al. 2018 *Human Molecular Genetics* doi: 10.1093/hmg/ddy429) [1].

Intriguingly, PMEL protein is a rarity in vertebrates: a **functional** amyloid. PMEL forms amyloid fibril structures in pigmented cells of vertebrates, including humans, and these amyloid fibrils are structurally critical for deposition of melanin pigment. The patient PMEL mutations we identified result in impaired processing of PMEL protein and altered amyloid structure, however the central question of how these changes lead to CNS neurodegeneration remains unknown. Our zebrafish modelling of PMEL loss via CRISPR/Cas9 mutation produced recessive inheritance of swollen eye morphology in larvae that is strikingly reminiscent of increased ocular pressure in glaucoma; this implies that loss of the functional PMEL amyloid can contribute to some aspects of PG aetiology. This is in contrast with our patient pedigrees and mutations of PMEL homologs in other animals (dogs, horses, etc.) where dominant inheritance implies a toxic gain-of-function; this predicts that abnormal PMEL amyloid might lead to neurodegeneration via a (prion-like?) templating of aberrant amyloid structure.

Future work is warranted to delineate these contributions of gained toxicity vs. reduced protein function during (prion-like?) neurodegeneration [2], including defining whether both of these contributions are

required for death of CNS neurons. Such work has potential to give unique insights into amyloidosis: We identify several mutations in patients with glaucomatous neurodegeneration, and these now guide our predictions of which aspects of PMEL are the key to human CNS cells thriving through a lifetime of high amyloid content.

**References**

[1] Lahola-Chomiak, et al. Hum Mol Genet. 2018. DOI:10.1093/hmg/ddy429

[2] Allison, et al. Int J Mol Sci. 2017;18:E2223. DOI:10.3390/ijms18102223

SCHMITT-ULMS, Gerold 2019

**236. A mechanism-based small-molecule approach to the treatment of prion diseases that targets $PrP^C$**

M Mehrabian<sup>a,b</sup>, D Williams<sup>a,b</sup>, X Wang<sup>a,b,c</sup>, F Ghodrati<sup>a,b,c</sup>, M Güneş,<sup>a,b</sup> and G Schmitt-Ulms<sup>a,b,c</sup>

<sup>a</sup>Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Ontario, Canada; <sup>b</sup>Krembil Discovery Centre, Toronto, Ontario, Canada; <sup>c</sup>Department of Laboratory Medicine & Pathobiology, University of Toronto, Toronto, Ontario, Canada

**ABSTRACT**

A wide range of observations in humans and animals indicate that a reduction in steady-state levels of the cellular prion protein ($PrP^C$) is both safe and would delay or prevent prion diseases. To derive rational approaches for suppressing $PrP^C$ levels at the plasma membrane, we and others have been studying the molecular environment and function of $PrP^C$.

This work led us to recognize that prion genes evolved from a ZIP zinc transporter ancestral molecule with roles in a morphogenetic programme known as epithelial-to-mesenchymal transition (EMT). The latter guided us to investigate the possible involvement of $PrP^C$ in EMT, revealing $PrP^C$ to control the polysialylation of the neural cell adhesion molecule 1 (NCAM1), a well-studied post-translational modification that serves as a molecular marker of EMT. In several cell models we interrogated, the molecular environment of $PrP^C$ is characterized by its residence within a specialized membrane domain that hosts, in addition to NCAM1, distinct subsets of more than 20 additional proteins. Interestingly, most of these proteins have known roles in the modulation of TGFB1 and integrin sister signalling complexes that are activated during EMT.

The binding of ligands to their cognate receptors often induces the internalization of ligand-receptor
<figure><img src="image_133.png" /></figure>

complexes, followed by their recycling or degradation. We reasoned that such a turn of events may lead to a passive co-internalization of PrP<sup>C</sup>, effectively reducing its levels at the cell surface. Our recent data reveal that PrP<sup>C</sup> levels are indeed robustly reduced in the presence of non-toxic nanomolar concentrations of a compound, hereafter referred to as KDC series 100 compound 2 (KDC102), which targets one of the proteins in proximity to PrP<sup>C</sup>. As anticipated, this reduction in PrP<sup>C</sup> levels depends on the compound-dependent degradation of the targeted receptor and can be observed in co-cultures of human neurons and astrocytes. Although mechanistic insights are not essential for translation into the clinic, their existence should prove an asset going forward. Current efforts are directed towards testing KDC102 in proof-of-concept prion disease treatment studies in rodents.

**237. A general mass spectrometry-based method of quantitating the relative amounts of PrP polymorphisms in CWD PrPSc**

Christopher J. Silva<sup>a</sup>, Melissa L. Erickson-Beltran<sup>b</sup>, Camilo Duque Velásquez<sup>c</sup>, Judd Aiken<sup>d</sup> and Debbie McKenzie<sup>e</sup>

<sup>a</sup>Produce Safety & Microbiology Research Unit USDA, WRRC, ARS;

<sup>b</sup>Produce Safety & Microbiology Research Unit, USDA, WRRC, ARS;

<sup>c</sup>University of Alberta, 120 Brain and Aging Research Building, Edmonton, AB T6G 2R3, Canada; <sup>d</sup>Agricultural Sciences - Centre for Prions and Protein Folding Diseases - University of Alberta;

<sup>e</sup>University of Alberta

**ABSTRACT**

**Background/Introduction:** Chronic wasting disease (CWD) was first described in a captive cervid herd in 1967, recognized as a prion disease in 1978, and first detected in wild cervids in 1981. CWD is readily transmitted among animals and from a contaminated environment to uninfected animals. By 2019, 26 states in the United States, three Canadian provinces, South Korea, Norway, and Finland had found CWD in captive and/or wild cervid populations. Cervid PrP contains polymorphisms that influence progression of CWD and propagation of CWD strains with different transmission properties [1-3]. It is important to develop a method to quantitate the relative amounts of those polymorphisms present in CWD PrP<sup>Sc</sup>, so that their respective influence on CWD prion propagation can be determined.

**Materials and Methods:** Plasmids containing relevant cervid PrP polymorphisms were prepared. The necessary <sup>15</sup>N-labelled internal standards were produced by

overexpressing the PrP genes in *E. coli* grown in minimal medium with <sup>15</sup>NH<sub>4</sub>Cl as the sole nitrogen source. Peptides derived from the tryptic or chymotryptic digestion of cervid recombinant PrP and containing the relevant polymorphisms were identified. A multiple reaction monitoring method (MRM) was developed for each of those peptides.

**Results:** Calibration curves relating the signal intensity of the various peptides and their absolute amounts as measure by protein assay were linear and had excellent correlation coefficients. In addition, samples from experimentally infected heterozygous deer (Q<sub>95</sub>S<sub>96</sub>/Q<sub>95</sub>G<sub>96</sub> and H<sub>95</sub>G<sub>96</sub> /Q<sub>95</sub>G<sub>96</sub>) were analysed and the relative amounts were determined. The optimal digestion for all tryptic peptides was 20 h. For chymotrypsin the optimal digestion time was also 20 h, apart from a single peptide.

**Conclusions:** This approach can be used to quantify the contribution of specific polymorphisms to PrP<sup>CWD</sup> formation, enhancing our ability to determine the relative susceptibility of various polymorphisms to CWD. It can also be used to detect the distribution of CWD prion strains.

**References**

[1] Johnson CJ, et al. PLoS One. 2011;6:e17450.

[2] Duque Velasquez C, et al. J Virol. 2015;89:12,362-12,373.

[3] Herbst A, et al. Emerg Infect Dis. 2017;23:1598-1600.

**238. A general mass spectrometry-based method of quantitating the oxidation of the methionine side chains in classical and atypical scrapie PrPSc**

Christopher J. Silva<sup>a</sup>, Melissa L. Erickson-Beltran<sup>b</sup>, Inmaculada Martin-Burriel<sup>c</sup>, Juan José Badiola<sup>d</sup>, Jesus R. Requena<sup>e</sup> and Rosa Bolea<sup>a,f</sup>

<sup>a</sup>Produce Safety & Microbiology Research Unit, USDA, WRRC, ARS;

<sup>b</sup>Produce Safety & Microbiology Research Unit, USDA, WRRC, ARS;

<sup>c</sup>University of Zaragoza, Zaragoza, Spain; <sup>d</sup>University of Zaragoza;

<sup>e</sup>University of Santiago de Compostela; <sup>f</sup>University of Zaragoza - Centre for TSE and emerging animal diseases

**ABSTRACT**

**Background/Introduction:** The structure of PrP<sup>Sc</sup> is uncertain, but the most compelling data suggests that it is a four-rung β-solenoid. The rungs of the solenoid are β-sheet secondary structures held together by hydrogen bonds. Unstructured loops connect the more rigid β-sheet structures. The side chains of the amino acids in the β-sheets alternately project inside
<figure><img src="image_137.png" /></figure>

complexes, followed by their recycling or degradation. We reasoned that such a turn of events may lead to a passive co-internalization of PrP<sup>C</sup>, effectively reducing its levels at the cell surface. Our recent data reveal that PrP<sup>C</sup> levels are indeed robustly reduced in the presence of non-toxic nanomolar concentrations of a compound, hereafter referred to as KDC series 100 compound 2 (KDC102), which targets one of the proteins in proximity to PrP<sup>C</sup>. As anticipated, this reduction in PrP<sup>C</sup> levels depends on the compound-dependent degradation of the targeted receptor and can be observed in co-cultures of human neurons and astrocytes. Although mechanistic insights are not essential for translation into the clinic, their existence should prove an asset going forward. Current efforts are directed towards testing KDC102 in proof-of-concept prion disease treatment studies in rodents.

**237. A general mass spectrometry-based method of quantitating the relative amounts of PrP polymorphisms in CWD PrP<Sc>**

Christopher J. Silva<sup>a</sup>, Melissa L. Erickson-Beltran<sup>b</sup>, Camilo Duque Velásquez<sup>c</sup>, Judd Aiken<sup>d</sup> and Debbie McKenzie<sup>e</sup>

<sup>a</sup>Produce Safety & Microbiology Research Unit USDA, WRRC, ARS;

<sup>b</sup>Produce Safety & Microbiology Research Unit, USDA, WRRC, ARS;

<sup>c</sup>University of Alberta, 120 Brain and Aging Research Building, Edmonton, AB T6G 2R3, Canada; <sup>d</sup>Agricultural Sciences - Centre for Prions and Protein Folding Diseases - University of Alberta;

<sup>e</sup>University of Alberta

**ABSTRACT**

**Background/Introduction:** Chronic wasting disease (CWD) was first described in a captive cervid herd in 1967, recognized as a prion disease in 1978, and first detected in wild cervids in 1981. CWD is readily transmitted among animals and from a contaminated environment to uninfected animals. By 2019, 26 states in the United States, three Canadian provinces, South Korea, Norway, and Finland had found CWD in captive and/or wild cervid populations. Cervid PrP contains polymorphisms that influence progression of CWD and propagation of CWD strains with different transmission properties [1-3]. It is important to develop a method to quantitate the relative amounts of those polymorphisms present in CWD PrP<sup>Sc</sup>, so that their respective influence on CWD prion propagation can be determined.

**Materials and Methods:** Plasmids containing relevant cervid PrP polymorphisms were prepared. The necessary <sup>15</sup>N-labelled internal standards were produced by

overexpressing the PrP genes in *E. coli* grown in minimal medium with <sup>15</sup>NH<sub>4</sub>Cl as the sole nitrogen source. Peptides derived from the tryptic or chymotryptic digestion of cervid recombinant PrP and containing the relevant polymorphisms were identified. A multiple reaction monitoring method (MRM) was developed for each of those peptides.

**Results:** Calibration curves relating the signal intensity of the various peptides and their absolute amounts as measure by protein assay were linear and had excellent correlation coefficients. In addition, samples from experimentally infected heterozygous deer (Q<sub>95</sub>S<sub>96</sub>/Q<sub>95</sub>G<sub>96</sub> and H<sub>95</sub>G<sub>96</sub> /Q<sub>95</sub>G<sub>96</sub>) were analysed and the relative amounts were determined. The optimal digestion for all tryptic peptides was 20 h. For chymotrypsin the optimal digestion time was also 20 h, apart from a single peptide.

**Conclusions:** This approach can be used to quantify the contribution of specific polymorphisms to PrP<sup>CWD</sup> formation, enhancing our ability to determine the relative susceptibility of various polymorphisms to CWD. It can also be used to detect the distribution of CWD prion strains.

**References**

[1] Johnson CJ, et al. PLoS One. 2011;6:e17450.

[2] Duque Velasquez C, et al. J Virol. 2015;89:12,362-12,373.

[3] Herbst A, et al. Emerg Infect Dis. 2017;23:1598-1600.

**238. A general mass spectrometry-based method of quantitating the oxidation of the methionine side chains in classical and atypical scrapie PrP<Sc>**

Christopher J. Silva<sup>a</sup>, Melissa L. Erickson-Beltran<sup>b</sup>, Inmaculada Martin-Burriel<sup>c</sup>, Juan José Badiola<sup>d</sup>, Jesus R. Requena<sup>e</sup> and Rosa Bolea<sup>a,f</sup>

<sup>a</sup>Produce Safety & Microbiology Research Unit, USDA, WRRC, ARS;

<sup>b</sup>Produce Safety & Microbiology Research Unit, USDA, WRRC, ARS;

<sup>c</sup>University of Zaragoza, Zaragoza, Spain; <sup>d</sup>University of Zaragoza;

<sup>e</sup>University of Santiago de Compostela; <sup>f</sup>University of Zaragoza - Centre for TSE and emerging animal diseases

**ABSTRACT**

**Background/Introduction:** The structure of PrP<sup>Sc</sup> is uncertain, but the most compelling data suggests that it is a four-rung β-solenoid. The rungs of the solenoid are β-sheet secondary structures held together by hydrogen bonds. Unstructured loops connect the more rigid β-sheet structures. The side chains of the amino acids in the β-sheets alternately project inside
the solenoid or outside of it. This may make the reactivity of adjacent amino side chains dramatically different. The side chain of methionine can be oxidized under physiological conditions to the much more polar sulfoxide. Eukaryotic cells possess enzymes that reduce oxidized methionines, so the methionines of $PrP^C$ should be largely unoxidized. Methionines present in $PrP^{Sc}$ may be oxidized by chemical reagents or natural oxidative processes. Quantitating the extent of this oxidation can provide insight into the location of the methionines within the $\beta$-solenoid.

**Materials and Methods:** Plasmids containing relevant PrP sequences were prepared.¹ ¹⁵N-labelled internal standards were prepared by overexpressing the PrP genes in *E. coli* grown in minimal medium with ¹⁵NH₄Cl as the sole nitrogen source. A set of peptides containing the methionines was identified that are derived from the tryptic or chymotryptic digestion of sheep PrP. A multiple reaction monitoring method (MRM) was developed to detect and quantitate each peptide.

**Results:** Calibration curves relating signal intensities of the various peptides and their absolute amounts were linear and had excellent correlation coefficients. The optimal digestion for all tryptic peptides was 20 h. For chymotrypsin the optimal digestion was 20 h, except for one peptide. In peptides containing two methionines, the oxidation state of each could be readily identified by a combination of chromatographic properties and the MRM method.

**Conclusions:** This approach can be used to quantitate the relative amounts of each oxidized methionine in sheep scrapie. By comparing the results obtained from classical and atypical scrapie, we gained insight into the structural differences between the two isoforms.

## 239. Hsp110 modifies prion infection in vitro and in vivo

Cristóbal Marrero-Winkens<sup>a,b,c</sup> and Hermann M. Schatzl<sup>a,b,c</sup>

**CONTACT** Cristóbal Marrero-Winkens cristobal.marrerowin@ucalgary.ca

**KEYWORDS:** Hsp110; chaperones; prion replication; prion fragmentation; disaggregase function

Mammalian heat shock protein 110 (Hsp110) family members cooperate with Hsp70 and Hsp40 to disaggregate misfolded proteins. Efficient *in vitro*

disaggregation of α-synuclein and artificially denatured proteins by this tri-chaperone system has recently been reported. In yeast, it is well established that the disaggregase formed by Hsp70 and Hsp104 (which has no mammalian homolog) affects the maintenance of prion phenotypes. We hypothesize that the mammalian tri-chaperone system fulfils a similar function by participating in prion fragmentation and disaggregation. To test this, Hsp110 levels were transiently manipulated in 22L-ScN2a cells. Transient knockdown reduced the levels of $PrP^{Sc}$, while overexpression led to a dose-dependent increase in $PrP^{Sc}$. These effects are reminiscent of Hsp104 modulation in yeast. Next, Hsp110 overexpressing and wild-type mice were inoculated with 22L and Me7 prions. Overexpression of Hsp110 significantly prolonged the survival of Me7- but not 22L-inoculated animals. Sucrose gradient ultracentrifugation of brain homogenates derived from terminally diseased animals also revealed a trend towards larger $PrP^{Sc}$ aggregates in overexpressing mice. Overall, these results confirm that Hsp110 is involved in $PrP^{Sc}$ fragmentation and warrant further study. Finally, we also report on on-going studies, which include prion inoculation of different overexpressing mice as well as the generation of Hsp110 knockout and overexpressing cell lines using the CRISPR-Cas9 system and recombinant lentiviruses

## 240. The NLRP3-Caspase 1 Negatively Regulates Autophagy via TLR4-TRIF in Prion Peptide-Infected Microglia

Mengyu Lait, Hao Yao†, Syed Zahid Ali Shah, Deming Zhao and Lifeng Yang

National Animal Transmissible Spongiform Encephalopathy Laboratory, College of Veterinary Medicine, China Agricultural University, Beijing, China

**CONTACT** Lifeng Yang yanglf2002@126.com

†Deceased.

## ABSTRACT

**Introduction:** Prion diseases are neurodegenerative disorders characterized by the accumulation of misfolded prion protein, spongiform changes in the brain, and brain inflammation as a result of the wide-spread activation of microglia. Autophagy is a highly conserved catabolic process for the clearance of cytoplasmic components, including protein aggregates and damaged organelles, this process also eliminates pathological $PrP^{Sc}$ as it accumulates during prion infection [1]. The NALP3 inflammasome is a multiprotein complex that is a component of the innate immune system and is responsible for the release of pro-

<footnote><sup>a</sup>Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, AB, Canada; <sup>b</sup>Calgary Prion Research Unit, AB, Canada; <sup>c</sup>Hotchkiss Brain Institute, University of Calgary, AB, Canada</footnote>
<figure><img src="image_138.png" /></figure>

the solenoid or outside of it. This may make the reactivity of adjacent amino side chains dramatically different. The side chain of methionine can be oxidized under physiological conditions to the much more polar sulfoxide. Eukaryotic cells possess enzymes that reduce oxidized methionines, so the methionines of $PrP^C$ should be largely unoxidized. Methionines present in $PrP^{Sc}$ may be oxidized by chemical reagents or natural oxidative processes. Quantitating the extent of this oxidation can provide insight into the location of the methionines within the $\beta$-solenoid.

**Materials and Methods:** Plasmids containing relevant PrP sequences were prepared.¹ ¹⁵N-labelled internal standards were prepared by overexpressing the PrP genes in *E. coli* grown in minimal medium with ¹⁵NH₄Cl as the sole nitrogen source. A set of peptides containing the methionines was identified that are derived from the tryptic or chymotryptic digestion of sheep PrP. A multiple reaction monitoring method (MRM) was developed to detect and quantitate each peptide.

**Results:** Calibration curves relating signal intensities of the various peptides and their absolute amounts were linear and had excellent correlation coefficients. The optimal digestion for all tryptic peptides was 20 h. For chymotrypsin the optimal digestion was 20 h, except for one peptide. In peptides containing two methionines, the oxidation state of each could be readily identified by a combination of chromatographic properties and the MRM method.

**Conclusions:** This approach can be used to quantitate the relative amounts of each oxidized methionine in sheep scrapie. By comparing the results obtained from classical and atypical scrapie, we gained insight into the structural differences between the two isoforms.

## 239. Hsp110 modifies prion infection in vitro and in vivo

Cristóbal Marrero-Winkens<sup>a,b,c</sup> and Hermann M. Schatzl<sup>a,b,c</sup>

**CONTACT** Cristóbal Marrero-Winkens cristobal.marrerowin@ucalgary.ca

**KEYWORDS:** Hsp110; chaperones; prion replication; prion fragmentation; disaggregase function

Mammalian heat shock protein 110 (Hsp110) family members cooperate with Hsp70 and Hsp40 to disaggregate misfolded proteins. Efficient *in vitro*

disaggregation of α-synuclein and artificially denatured proteins by this tri-chaperone system has recently been reported. In yeast, it is well established that the disaggregase formed by Hsp70 and Hsp104 (which has no mammalian homolog) affects the maintenance of prion phenotypes. We hypothesize that the mammalian tri-chaperone system fulfils a similar function by participating in prion fragmentation and disaggregation. To test this, Hsp110 levels were transiently manipulated in 22L-ScN2a cells. Transient knockdown reduced the levels of $PrP^{Sc}$, while overexpression led to a dose-dependent increase in $PrP^{Sc}$. These effects are reminiscent of Hsp104 modulation in yeast. Next, Hsp110 overexpressing and wild-type mice were inoculated with 22L and Me7 prions. Overexpression of Hsp110 significantly prolonged the survival of Me7- but not 22L-inoculated animals. Sucrose gradient ultracentrifugation of brain homogenates derived from terminally diseased animals also revealed a trend towards larger $PrP^{Sc}$ aggregates in overexpressing mice. Overall, these results confirm that Hsp110 is involved in $PrP^{Sc}$ fragmentation and warrant further study. Finally, we also report on on-going studies, which include prion inoculation of different overexpressing mice as well as the generation of Hsp110 knockout and overexpressing cell lines using the CRISPR-Cas9 system and recombinant lentiviruses

## 240. The NLRP3-Caspase 1 Negatively Regulates Autophagy via TLR4-TRIF in Prion Peptide-Infected Microglia

Mengyu Lait, Hao Yao†, Syed Zahid Ali Shah, Deming Zhao and Lifeng Yang

National Animal Transmissible Spongiform Encephalopathy Laboratory, College of Veterinary Medicine, China Agricultural University, Beijing, China

**CONTACT** Lifeng Yang yanglf2002@126.com

†Deceased.

## ABSTRACT

**Introduction:** Prion diseases are neurodegenerative disorders characterized by the accumulation of misfolded prion protein, spongiform changes in the brain, and brain inflammation as a result of the wide-spread activation of microglia. Autophagy is a highly conserved catabolic process for the clearance of cytoplasmic components, including protein aggregates and damaged organelles, this process also eliminates pathological $PrP^{Sc}$ as it accumulates during prion infection [1]. The NALP3 inflammasome is a multiprotein complex that is a component of the innate immune system and is responsible for the release of pro-

<footnote><sup>a</sup>Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, AB, Canada; <sup>b</sup>Calgary Prion Research Unit, AB, Canada; <sup>c</sup>Hotchkiss Brain Institute, University of Calgary, AB, Canada</footnote>
<figure><img src="image_134.png" /></figure>

inflammatory cytokines. Our previous study showed that the neurotoxic prion peptide PrP106-126 induces NALP3 inflammasome activation and subsequent IL-1β release in microglia [2]. Autophagy is involved in the regulation of the immune responses and inflammation in many diseases including neurodegenerative diseases. However, the relationship between autophagy and NALP3 inflammasome in prion diseases has not been investigated.

**Results:** In this study, we demonstrated that the processing and release of mature IL-1β is significantly enhanced by the inhibition of autophagy. Conversely, gene silencing of the NALP3 inflammasome promotes autophagy. Suppression of TRIF or TLR4 by siRNA attenuated PrP106-126-induced autophagy, which is indicating that the TLR4-TRIF signalling pathway is involved in PrP106-26-induced autophagy. Caspase 1 directly cleaved TRIF to diminish TLR-4-TRIF mediated autophagy. Our findings suggest that the inhibition of autophagy by NALP3 inflammasome is probably mediated by activated Caspase-1-induced TRIF cleavage [3]. This is the first study reporting that the NALP3 inflammasome complex negatively regulates autophagy in response to PrP106-126 stimulation in microglia, and partly explains the mechanism of autophagy inhibition by Caspase-1 in PrP106-126-induced BV2 cell activation.

**Conclusions:** Our findings suggest that autophagy upregulation and inhibition of Caspase-1 may protect against prion-induced neuroinflammation and accelerate misfolded protein degradation and are potential therapeutic approaches for prion diseases.

## References

[1] Deretic V, et al. Autophagy in infection, inflammation and immunity. Nat Rev Immunol 2013;13:722-737.

[2] Shi F, et al. Inhibition of phagocytosis and lysosomal acidification suppresses neurotoxic prion peptide induced NALP3 inflammasome activation in BV2 microglia. J Neuroimmunol. 2013;260:121-125.

[3] Faure M., et al. Pathogen-induced autophagy signaling in innate immunity. J Innate Immun. 2013;5:456-470.

## 241. Evaluating the applicability of tauK18 as RT-QuIC reaction substrate for the analysis of samples collected from patients with neurodegenerative disorders

Martina Rossiª, Chiara Maria Giulia De Lucaª, Elena De Ceccoª, Joanna Narkiewiczª, Giulia Salzanoª, Sara Maria Portaleoneᵇ, Antonio Emanuele Eliaᶜ, Roberto Eleopraᶜ, Giuseppe Legnameª, Giorgio Giacconeᵈ and Fabio Modaᵈ

ªLaboratory of Prion Biology, Department of Neuroscience, Scuola Internazionale Superiore di Studi Avanzati, Trieste, Italy;
ᵇNeuropathology-Neurology 5 Unit, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy;
ᶜMovement Disorders Department – Neurology I Unit, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milano, Italy;
ᵈOtolaryngology Unit, San Paolo Hospital, Department of Health Sciences, University of Milan, Milan, Italy

CONTACT Martina Rossi martina.rossi@sissa.it

## ABSTRACT

**Introduction:** Neurodegenerative diseases are characterized by the accumulation of abnormally folded proteins in different regions of the brain [1,2]. Among them, Alzheimer's disease (AD), Frontotemporal dementia (FTLD-tau), Corticobasal Degeneration (CBD), and Progressive Supranuclear Palsy (PSP) are associated to different abnormal conformations of the microtubule-associated tau protein. Clinical diagnosis of tauopathies is often challenging due to their overlapping symptoms, especially in the early stages [2]. Compelling evidence suggest that abnormally folded proteins appear in peripheral tissues of demented patients [3-5]. Therefore, we are currently exploiting the Real-Time Quaking Induced Conversion (RT-QuIC) assay to attempt detection of abnormal tau in peripheral tissues of patients with tauopathies.

**Materials and methods:** Recombinant tau fragment comprising the 4-repeats domain (tauK18) was used as substrate for RT-QuIC assay. *In vitro* produced tauK18 aggregates (referred to as artificial seeds) were serially diluted and added to new RT-QuIC reactions to assess its sensitivity. Similarly, brain homogenates (BHs) collected from patients with PSP, FTLD-tau and AD were analysed by means of RT-QuIC. BHs collected from patients with other dementias (including Parkinson's disease, Dementia with Lewy bodies and Multiple System Atrophy) and subject not affected by neurodegenerative disorders (NDP) were used as controls. Olfactory mucosa (OM) samples collected from patients with a clinical diagnosis of CBD, PSP, and AD are currently under RT-QuIC analysis. OM from PD, MSA, DLB patients, and healthy subjects are included as controls.

**Results:** All dilutions of artificial seed efficiently accelerated the kinetics of tauK18 aggregation, thus revealing the ability of RT-QuIC in detecting up to attograms of tauK18 aggregates. BHs of patients with PSP, FTLD-tau and AD accelerated tauK18 aggregation with greater efficiency than that of DLB, PD, MSA, and NDP. Preliminary data collected from RT-QuIC analysis of OM samples suggest that the aggregation of
<figure><img src="image_139.png" /></figure>

inflammatory cytokines. Our previous study showed that the neurotoxic prion peptide PrP106-126 induces NALP3 inflammasome activation and subsequent IL-1β release in microglia [2]. Autophagy is involved in the regulation of the immune responses and inflammation in many diseases including neurodegenerative diseases. However, the relationship between autophagy and NALP3 inflammasome in prion diseases has not been investigated.

**Results:** In this study, we demonstrated that the processing and release of mature IL-1β is significantly enhanced by the inhibition of autophagy. Conversely, gene silencing of the NALP3 inflammasome promotes autophagy. Suppression of TRIF or TLR4 by siRNA attenuated PrP106-126-induced autophagy, which is indicating that the TLR4-TRIF signalling pathway is involved in PrP106-26-induced autophagy. Caspase 1 directly cleaved TRIF to diminish TLR-4-TRIF mediated autophagy. Our findings suggest that the inhibition of autophagy by NALP3 inflammasome is probably mediated by activated Caspase-1-induced TRIF cleavage [3]. This is the first study reporting that the NALP3 inflammasome complex negatively regulates autophagy in response to PrP106-126 stimulation in microglia, and partly explains the mechanism of autophagy inhibition by Caspase-1 in PrP106-126-induced BV2 cell activation.

**Conclusions:** Our findings suggest that autophagy upregulation and inhibition of Caspase-1 may protect against prion-induced neuroinflammation and accelerate misfolded protein degradation and are potential therapeutic approaches for prion diseases.

## References

[1] Deretic V, et al. Autophagy in infection, inflammation and immunity. Nat Rev Immunol 2013;13:722-737.

[2] Shi F, et al. Inhibition of phagocytosis and lysosomal acidification suppresses neurotoxic prion peptide induced NALP3 inflammasome activation in BV2 microglia. J Neuroimmunol. 2013;260:121-125.

[3] Faure M., et al. Pathogen-induced autophagy signaling in innate immunity. J Innate Immun. 2013;5:456-470.

# 241. Evaluating the applicability of tauK18 as RT-QuIC reaction substrate for the analysis of samples collected from patients with neurodegenerative disorders

Martina Rossiª, Chiara Maria Giulia De Lucaª, Elena De Ceccoª, Joanna Narkiewiczª, Giulia Salzanoª, Sara Maria Portaleoneᵇ, Antonio Emanuele Eliaᶜ, Roberto Eleopraᶜ, Giuseppe Legnameª, Giorgio Giacconeᵈ and Fabio Modaᵈ

ªLaboratory of Prion Biology, Department of Neuroscience, Scuola Internazionale Superiore di Studi Avanzati, Trieste, Italy;
ᵇNeuropathology-Neurology 5 Unit, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy;
ᶜMovement Disorders Department – Neurology I Unit, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milano, Italy;
ᵈOtolaryngology Unit, San Paolo Hospital, Department of Health Sciences, University of Milan, Milan, Italy

CONTACT Martina Rossi martina.rossi@sissa.it

## ABSTRACT

**Introduction:** Neurodegenerative diseases are characterized by the accumulation of abnormally folded proteins in different regions of the brain [1,2]. Among them, Alzheimer's disease (AD), Frontotemporal dementia (FTLD-tau), Corticobasal Degeneration (CBD), and Progressive Supranuclear Palsy (PSP) are associated to different abnormal conformations of the microtubule-associated tau protein. Clinical diagnosis of tauopathies is often challenging due to their overlapping symptoms, especially in the early stages [2]. Compelling evidence suggest that abnormally folded proteins appear in peripheral tissues of demented patients [3-5]. Therefore, we are currently exploiting the Real-Time Quaking Induced Conversion (RT-QuIC) assay to attempt detection of abnormal tau in peripheral tissues of patients with tauopathies.

**Materials and methods:** Recombinant tau fragment comprising the 4-repeats domain (tauK18) was used as substrate for RT-QuIC assay. *In vitro* produced tauK18 aggregates (referred to as artificial seeds) were serially diluted and added to new RT-QuIC reactions to assess its sensitivity. Similarly, brain homogenates (BHs) collected from patients with PSP, FTLD-tau and AD were analysed by means of RT-QuIC. BHs collected from patients with other dementias (including Parkinson's disease, Dementia with Lewy bodies and Multiple System Atrophy) and subject not affected by neurodegenerative disorders (NDP) were used as controls. Olfactory mucosa (OM) samples collected from patients with a clinical diagnosis of CBD, PSP, and AD are currently under RT-QuIC analysis. OM from PD, MSA, DLB patients, and healthy subjects are included as controls.

**Results:** All dilutions of artificial seed efficiently accelerated the kinetics of tauK18 aggregation, thus revealing the ability of RT-QuIC in detecting up to attograms of tauK18 aggregates. BHs of patients with PSP, FTLD-tau and AD accelerated tauK18 aggregation with greater efficiency than that of DLB, PD, MSA, and NDP. Preliminary data collected from RT-QuIC analysis of OM samples suggest that the aggregation of
<figure><img src="image_135.png" /></figure>

tauK18 is more efficient when the reaction is supplemented with CBD and PSP samples. Less efficient aggregation was observed after the addition of the other OM samples.

**Conclusions:** The aggregation kinetics of tauK18 used as RT-QuIC reaction substrate can be influenced by other factors other than tau itself, therefore representing an important issue especially from a diagnostic point of view. The presence of other amyloidogenic proteins (e.g. α-synuclein or β-amyloid) can play a pivotal role in promoting tauK18 aggregation, thus generating misleading results. For this reason, we are currently coupling RT-QuIC analysis with structural and biochemical studies to assess whether the final reaction products acquire peculiar features useful for disease discrimination.

**KEYWORDS:** tauK18; RT-QuIC; diagnosis; tauopathies

## References

[1] Soto C. Nat Rev Neurosci. 2003;4:49.

[2] Irwin DJ. Parkinsonism Relat Disord. 2015;22 Suppl 1 (01):S29-S33.

[3] Moda F. et al., N Engl J Med. 7 August 2014;371 (6):530-539.

[4] Saijo E. et al., Acta Neuropathol. 2017 May;133(5):751-765.

[5] Redaelli V, Bistaffa E. et al., Sci Rep. 7 April 2017;7:46269.

## 242. Incubation periods of classical and atypical bovine spongiform encephalopathies (BSE) reflect an inverse relationship between PrP<sup>Sc</sup>-associated neuroinflammation and autophagy

Najiba Mammadova<sup>a,b,c</sup>, M. Heather West Greenlee<sup>b,c,d</sup>, S. Jo Moore<sup>e</sup>, Donald S. Sakaguchi<sup>a,c</sup> and Justin J. Greenlee<sup>e</sup>

CONTACT M. Heather West Greenlee mheather@iastate.edu

## ABSTRACT

Neurodegenerative protein misfolding disorders are a group of diseases in both humans and animals with a similar mechanism of disease progression. These diseases, that include Alzheimer's disease, Parkinson's disease, chronic wasting disease, and bovine

spongiform encephalopathy (BSE) result from aberrant folding and accumulation of disease specific proteins. Transmissible spongiform encephalopathies (TSEs) are diseases that result from improper folding and accumulation of the prion protein. Due to their transmissibility, TSEs are an important experimental model to study the basic pathobiology of all protein misfolding disorders. TSE strain variations can influence disease phenotypes such as host susceptibility, biochemical and immunohistochemical profiles, and incubation periods. BSE is a TSE that occurs in cattle that can be subdivided into three different strains: classical BSE, atypical high (H)-type, and atypical low (L)-type BSE. Both H-type and L-type BSEs, have shorter incubation periods after experimental inoculation and, therefore, an accelerated disease progression when compared to classical BSE. Currently, there is a lack of knowledge about the factors that influence accumulation of misfolded proteins and disease progression making this a key challenge for the development of therapies for protein misfolding diseases. In this study, we used the differences between classical (n = 12) and atypical BSEs (n = 20) as a model to identify the molecular factors associated with disease progression. The NLRP3 inflammasome is a critical component of the innate immune system that leads to release of IL-1β (Interlukin-1β), an important regulator of neuroinflammation in many protein misfolding diseases. Macroautophagy is an intracellular mechanism that plays an essential role in protein clearance and homeostasis. Here we used immunohistochemistry to assess the retina and three brain regions (thalamus, caudate nucleus, and brainstem at the level of the obex) to investigate the relationship between disease incubation period, PrP<sup>Sc</sup> accumulation, neuroinflammation, and changes in macroautophagy. We demonstrate that atypical BSEs, characterized by shorter incubation periods, present with greater accumulation of PrP<sup>Sc</sup>, glial-cell activation, NLRP3 inflammasome activation, and decreased autophagy. Our work demonstrates a relationship between disease time course, neuroinflammation, and the autophagic stress response, that has not been previously reported. This work may help identify novel therapeutic approaches that can delay or even prevent the progression of protein-misfolding diseases.

## 243. Comparative effect of green tea polyphenols on the amyloid aggregation of human prion protein

Nikita Admane and Abhinav Grover

<footnote><sup>a</sup>Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA, USA; <sup>b</sup>Immunobiology Graduate Program, Iowa State University, Ames, IA, USA; <sup>c</sup>Neuroscience Graduate Program, Iowa State University, Ames, IA, USA; <sup>d</sup>Department of Biomedical Sciences, Iowa State University College of Veterinary Medicine, Ames, IA, USA; <sup>e</sup>Virus and Prion Research Unit, National Animal Disease Center, USDA, Agricultural Research Service, Ames, IA, USA</footnote>
tauK18 is more efficient when the reaction is supplemented with CBD and PSP samples. Less efficient aggregation was observed after the addition of the other OM samples.

**Conclusions:** The aggregation kinetics of tauK18 used as RT-QuIC reaction substrate can be influenced by other factors other than tau itself, therefore representing an important issue especially from a diagnostic point of view. The presence of other amyloidogenic proteins (e.g. α-synuclein or β-amyloid) can play a pivotal role in promoting tauK18 aggregation, thus generating misleading results. For this reason, we are currently coupling RT-QuIC analysis with structural and biochemical studies to assess whether the final reaction products acquire peculiar features useful for disease discrimination.

**KEYWORDS:** tauK18; RT-QuIC; diagnosis; tauopathies

## References

[1] Soto C. Nat Rev Neurosci. 2003;4:49.

[2] Irwin DJ. Parkinsonism Relat Disord. 2015;22 Suppl 1 (01):S29-S33.

[3] Moda F. et al., N Engl J Med. 7 August 2014;371 (6):530-539.

[4] Saijo E. et al., Acta Neuropathol. 2017 May;133(5):751-765.

[5] Redaelli V, Bistaffa E. et al., Sci Rep. 7 April 2017;7:46269.

# 242. Incubation periods of classical and atypical bovine spongiform encephalopathies (BSE) reflect an inverse relationship between PrP<sup>Sc</sup>-associated neuroinflammation and autophagy

Najiba Mammadova<sup>a,b,c</sup>, M. Heather West Greenlee<sup>b,c,d</sup>, S. Jo Moore<sup>e</sup>, Donald S. Sakaguchi<sup>a,c</sup> and Justin J. Greenlee<sup>e</sup>

CONTACT M. Heather West Greenlee mheather@iastate.edu

## ABSTRACT

Neurodegenerative protein misfolding disorders are a group of diseases in both humans and animals with a similar mechanism of disease progression. These diseases, that include Alzheimer's disease, Parkinson's disease, chronic wasting disease, and bovine

spongiform encephalopathy (BSE) result from aberrant folding and accumulation of disease specific proteins. Transmissible spongiform encephalopathies (TSEs) are diseases that result from improper folding and accumulation of the prion protein. Due to their transmissibility, TSEs are an important experimental model to study the basic pathobiology of all protein misfolding disorders. TSE strain variations can influence disease phenotypes such as host susceptibility, biochemical and immunohistochemical profiles, and incubation periods. BSE is a TSE that occurs in cattle that can be subdivided into three different strains: classical BSE, atypical high (H)-type, and atypical low (L)-type BSE. Both H-type and L-type BSEs, have shorter incubation periods after experimental inoculation and, therefore, an accelerated disease progression when compared to classical BSE. Currently, there is a lack of knowledge about the factors that influence accumulation of misfolded proteins and disease progression making this a key challenge for the development of therapies for protein misfolding diseases. In this study, we used the differences between classical (n = 12) and atypical BSEs (n = 20) as a model to identify the molecular factors associated with disease progression. The NLRP3 inflammasome is a critical component of the innate immune system that leads to release of IL-1β (Interlukin-1β), an important regulator of neuroinflammation in many protein misfolding diseases. Macroautophagy is an intracellular mechanism that plays an essential role in protein clearance and homeostasis. Here we used immunohistochemistry to assess the retina and three brain regions (thalamus, caudate nucleus, and brainstem at the level of the obex) to investigate the relationship between disease incubation period, PrP<sup>Sc</sup> accumulation, neuroinflammation, and changes in macroautophagy. We demonstrate that atypical BSEs, characterized by shorter incubation periods, present with greater accumulation of PrP<sup>Sc</sup>, glial-cell activation, NLRP3 inflammasome activation, and decreased autophagy. Our work demonstrates a relationship between disease time course, neuroinflammation, and the autophagic stress response, that has not been previously reported. This work may help identify novel therapeutic approaches that can delay or even prevent the progression of protein-misfolding diseases.

# 243. Comparative effect of green tea polyphenols on the amyloid aggregation of human prion protein

Nikita Admane and Abhinav Grover

<footnote><sup>a</sup>Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA, USA; <sup>b</sup>Immunobiology Graduate Program, Iowa State University, Ames, IA, USA; <sup>c</sup>Neuroscience Graduate Program, Iowa State University, Ames, IA, USA; <sup>d</sup>Department of Biomedical Sciences, Iowa State University College of Veterinary Medicine, Ames, IA, USA; <sup>e</sup>Virus and Prion Research Unit, National Animal Disease Center, USDA, Agricultural Research Service, Ames, IA, USA</footnote>