Document text
Principal Investigator: SUZETTE Alise PRIOLA
Organization: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
Fiscal Year: 2020
Award: $1,145,487
Funding agency: National Institute of Allergy and Infectious Diseases
Transmissible spongiform encephalopathies (TSEs or prion diseases) are a group of rare neurodegenerative diseases which include sporadic Creutzfeldt-Jakob disease (sCJD) in humans, scrapie in sheep, bovine spongiform encephalopathy (BSE), and chronic wasting disease (CWD) in mule deer and elk. Prions can cross species barriers. The fact that BSE has infected humans in Great Britain and concerns that CWD may act similarly in the US underscores the importance of understanding prion pathogenesis and developing effective therapeutics. The infectious agent of prion diseases is called a prion and is largely composed of an abnormally refolded, protease resistant form (PrPSc) of the normal, protease-sensitive prion protein, PrPC. Susceptibility to infection can be influenced by amino acid homology between PrPC and PrPSc while differences in structure between PrPSc molecules are believed to encode strain phenotypes. My laboratory addresses different aspects of prion diseases at both the molecular and pathogenic level including: 1) identifying the earliest events which occur during prion infection, 2) defining the molecular pathways involved in prion-associated neurodegeneration, 3) determining the molecular basis of prion strains, 4) determining how PrPC sequence and post-translational modifications influence PrPSc formation and disease phenotype and, 5) development of effective prion therapeutics.
Although there is an increasing body of work suggesting that mitochondrial dysfunction is important in several neurodegenerative protein misfolding diseases such as Alzheimers disease (AD) and Parkinsons disease (PD), the role of mitochondria in prion disease is poorly understood. We have found that mitochondrial pathways of apoptosis are implicated in non-amyloid forms of prion disease (Moore et al. J. Proteome Res. 13: 4620 (2014), Annual Report 2014) and were the first to show that mitochondrial respiration is impaired in late stage prion disease (Faris et al., J. Virol. 91: e00524-17 (2017), Annual Report 2017). We have also published data showing that PrPC is present in brain mitochondria from healthy wild-type and transgenic mice (Faris et al., Sci. Rep. 7: 41556 (2017), Annual Report 2017) suggesting that, as has been proposed for other proteins associated with neurodegenerative disorders, PrPC may play a role in mitochondrial function.
In 2020, we continued studies looking at prion disease progression and mitochondrial dysfunction using mice with known mitochondrial defects. These studies are part of a collaboration with Dr. Catharine Bosios laboratory which has a Seahorse XF Analyzer to measure mitochondrial respiration and viability. We have found that, in mice in which a gene involved in axonal degeneration has been knocked out, more rapid prion disease progression is associated with an unexpected increase in mitochondrial respiration. We are currently investigating the basis of this increase in mitochondrial oxygen consumption.
In 2020 we inoculated prions into three different lines of transgenic mice, each of which had a different gene involved in mitochondrial dynamics ablated. Our results show that prion disease incubation times are differentially affected depending upon the gene that has been knocked out. We are in the process of analyzing mitochondrial function in these mice in order to determine how alterations in mitochondrial dynamics may influence prion disease progression.
In 2020 post-doctoral fellow Dr. Daniel Shoup began testing how cell lines he derived in 2019, which express a reporter gene to measure mitochondrial redox stress, responded to exposure to prions. His preliminary results suggest that prion infection affects the redox state of the cell in a prion-strain dependent manner. These studies are ongoing, and the results will provide insights into the mechanisms involved in cellular loss during prion infection.
Amino acid mismatches between PrPC and PrPSc influence the rate at which PrPSc forms. Thus, heterozygosity at key residues has the potential to significantly slow or even prevent disease transmission (J Gen Virol 93: 2749-2756 (2012)). In human PrPC, methionine/valine heterozygosity at codon 129 influences prion transmission and is a known resistance factor to sCJD (ACTA Neuropathol 130: 159-170 (2015)). We hypothesized that the PrPSc allotype ratio, i.e. the ratio of PrPSc with methionine at codon 129 (PrPSc-M129) to PrPSc with valine at codon 129 (PrPSc-V129), determines how efficiently prions from heterozygous cases of CJD transmit disease. Consistent with the possibility that PrPSc allotype could impact disease transmission, we found that the PrPSc allotype ratio varied in codon 129 MV heterozygous cases of sCJD (Moore et al. PLoS Pathog. 12: e1005416 (2016), Annual Report 2016), with some cases having a greater amount of PrPSc-M129 than others. Based on these data, we further hypothesized that those cases with higher levels of PrPSc-M129 would more efficiently transmit disease to mice expressing human PrPC with a methionine at codon 129 (PrPC-M129).
In 2020, we published the results of transmission of 10 cases of codon 129 MV heterozygous CJD into two different lines of susceptible transgenic mice overexpressing PrPC-M129. Our data showed that, while PrPSc allotype was not predictive of disease incubation time, it did appear to correlate with the type of PrPSc deposited. Our data also showed that, within a single brain, prions with different infectious properties were present in brain regions with different PrPSc allotypes. This latter observation may help to explain why multiple prion strains can be isolated from a single brain. Cumulatively, our data demonstrated that there was more heterogeneity in the transmission properties of CJD neurological subtypes than has been previously described and suggest a complex picture of CJD transmission where CJD subtype, PrPSc conformation, PrPSc allotype, and the host likely all contribute to the final disease phenotype.
The prion agent is notoriously difficult to inactivate with the routine sterilization protocols used in hospitals where iatrogenic transmission of CJD is an ongoing concern. The extreme resistance of prions to inactivation and their ability to persist in the environment for decades thus remain significant public health issues. Similar concerns apply to the laboratory setting. It is often necessary to analyze prion samples using advanced analytical techniques that are frequently only available outside of biosafety level 2 (BSL-2) containment, the minimum biosafety level required for studying infectious prions. However, the remarkable resistance of prions to inactivation can make it difficult to produce and analyze prion samples free of infectivity that still retain sufficient sample integrity for research purposes. We recently published a study demonstrating that a straightforward denaturation and in-gel protease digestion protocol used to prepare prion-infected samples for mass spectroscopy leads to the loss of at least 7 logs of prion infectivity (Moore et al., Biochim Biophys Acta Proteins Proteom. 1866: 1174 (2018), Annual Report 2018). In 2020, we continued in vivo studies to determine the amount of prion infectivity in CJD samples that were processed for mass spectrometry using several other common methods. Additionally, we expanded these studies in 2020 to include hamster prions. Hamster prion titers are 4-5 logs higher than in humans and will enable us to determine if the mass spectrometry techniques used inactivate very high titer prions. These experiments will take at least another year to complete but the results will be of use to regulators, biosafety specialists, and researchers tasked with determining whether or not prion-infected samples can be safely analyzed outside of BSL-2 containment.
Terms: <AD dementia><Acute><Address><Affect><Alzheimer><Alzheimer Type Dementia><Alzheimer disease><Alzheimer sclerosis><Alzheimer syndrome><Alzheimer's><Alzheimer's Disease><Alzheimer's disease dementia><Alzheimers Dementia><Alzheimers disease><Amino Acids><Annual Reports><Apoptosis><Apoptosis Pathway><Biochemical><Bovine Spongiform Encephalitis><Bovine Spongiform Encephalopathy><Brain><Brain Nervous System><Brain region><British Isles><CJD><Cell Body><Cell Line><CellLine><Cells><Chronic Wasting Disease><Codon><Codon Nucleotides><Collaborations><Complex><Containment><Creutzfeldt-Jacob Disease><Creutzfeldt-Jakob Disease><Creutzfeldt-Jakob Syndrome><Cricetinae><Data><Deer><Defect><Degenerative Neurologic Diseases><Degenerative Neurologic Disorders><Deposit><Deposition><Development><Digestion><Disease><Disease Progression><Disorder><Encephalon><Environment><Equine mule><Esteroproteases><Event><Exposure to><Gel><Genes><Genus Hippocampus><Great Britain><Hamsters><Hamsters Mammals><Heterogeneity><Heterozygote><Hospitals><Human><Iatrogenesis><Impairment><Infection><Infectious Agent><Investigators><Jakob-Creutzfeldt Disease><Knock-out><Knockout><L-Valine><Laboratories><Mad Cow Disease><Mass Photometry/Spectrum Analysis><Mass Spectrometry><Mass Spectroscopy><Mass Spectrum><Mass Spectrum Analyses><Mass Spectrum Analysis><Measures><Methionine><Methods><Mice><Mice Mammals><Mitochondria><Modern Man><Molecular><Molecular Configuration><Molecular Conformation><Molecular Genetics><Molecular Stereochemistry><Mule><Murine><Mus><Nerve Degeneration><Nervous System Degenerative Diseases><Neural Degenerative Diseases><Neural degenerative Disorders><Neurodegenerative Diseases><Neurodegenerative Disorders><Neurologic><Neurologic Degenerative Conditions><Neurological><Neuron Degeneration><Ovis><Oxidation-Reduction><Oxygen Consumption><Paralysis Agitans><Parkinson><Parkinson Disease><Parkinson's disease><Parkinsons disease><Pathogenesis><Pathogenicity><Pathway interactions><Peptidases><Peptide Hydrolases><Phenotype><Play><Post-Translational Modification Protein/Amino Acid Biochemistry><Post-Translational Modifications><Post-Translational Protein Modification><Post-Translational Protein Processing><Postdoc><Postdoctoral Fellow><Posttranslational Modifications><Posttranslational Protein Processing><PrP><PrP Proteins><PrPSc><PrPSc Proteins><Predisposition><Primary Parkinsonism><Primary Senile Degenerative Dementia><Prion Diseases><Prion Protein Diseases><Prion Proteins><Prion-Induced Disorder><Prions><Process><Programmed Cell Death><Property><Prophylactic treatment><Prophylaxis><Protease Gene><Proteases><Protein Modification><Proteinases><Proteins><Proteolytic Enzymes><Proteome><Protocol><Protocols documentation><Public Health><Publishing><R Plasmids><Redox><Reporter Genes><Research><Research Associate><Research Personnel><Researchers><Resistance><Respiration><Role><Sampling><Scrapie><Scrapie PrP><Seahorse><Sheep><Specialist><Sterilization><Strains Cell Lines><Stress><Structure><Subacute Spongiform Encephalopathy><Susceptibility><Techniques><Testing><Therapeutic><Time><Transgenic Mice><Transmissible Dementias><Transmissible Spongiform Encephalopathies><Transmission><Treatment Efficacy><VAC-TX><Vaccine Therapy><Valine><Wild Type Mouse><aberrant protein folding><abnormal protein folding><advanced analytics><aminoacid><axon degeneration><axonal degeneration><base><communicable disease transmission><conformation><conformational state><cultured cell line><degenerative axon><degenerative diseases of motor and sensory neurons><degenerative neurological diseases><dementia of the Alzheimer type><developmental><disease phenotype><disease transmission><disease-associated PrP><experiment><experimental research><experimental study><heterozygosity><iatrogenic><iatrogenically><iatrogenicity><in vivo><infectious disease transmission><infectious organism><insight><intervention efficacy><mitochondrial><mitochondrial dysfunction><neural degeneration><neurodegeneration><neurodegenerative><neurodegenerative illness><neurological degeneration><neuronal degeneration><overexpress><overexpression><oxidation reduction reaction><pathologic protein folding><pathway><post-doc><post-doctoral><prevent><preventing><primary degenerative dementia><prion disorder><protein mis-folding><protein misfolding><resistance factors><resistant><respiratory mechanism><senile dementia of the Alzheimer type><social role><spongiform degeneration><spongiform encephalopathy><therapeutic efficacy><therapeutic vaccination><therapeutically effective><therapy efficacy><transmission process><wildtype mouse>