Molecular Mechanisms of Prion Protein Amyloid Formation

National Institute Of Allergy And Infectious Diseases

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Abstract

Transmissible spongiform encephalopathies (TSEs or prion diseases) are a group of rare neurodegenerative diseases which include scrapie in sheep, bovine spongiform encephalopathy (BSE), and chronic wasting disease (CWD) in mule deer and elk. In humans, the most common type of prion disease is Creutzfeldt-Jakob disease (CJD) which can occur in several forms. Sporadic CJD (sCJD) makes up the majority of CJD cases and occurs randomly at an incidence of 1-2 per million people worldwide. Iatrogenic CJD (iCJD) is associated with exposure to prion contaminated medical instruments or products while familial CJD (fCJD) is associated with mutations in the prion protein gene. The infectious agent of TSE 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. PrPSc can be deposited in the brain as either diffuse amyloid negative deposits or as dense amyloid positive deposits. For reasons that are not yet clear, amyloid forms of prion disease appear to be less transmissible than non-amyloid forms. Furthermore, it is unknown if prion diseases where PrPSc is deposited primarily as amyloid follow the same pathogenic processes as prion diseases where PrPSc is primarily deposited as non-amyloid. Multiple studies have shown that amyloid formed from amyloid beta (A) protein, alpha synuclein and tau also propagate via prion-like mechanisms and spread from cell-to-cell in transgenic mouse models (e.g. Science 313: 1781-1784 (2006), Nat Cell Biol 11: 909-913 (2009), J Exp Med 209: 975-986 (2012)). Based on these data, it has been suggested that amyloid formation in neurodegenerative proteinopathies such as Alzheimers Disease (AD) and Parkinsons disease (PD) occurs via prion-like mechanisms and that proteins such as AD-associated A may also be transmissible, infectious prions. Co-deposition of misfolded proteins during neurodegeneration, such as the co-localization of PrPSc and A to plaques in some cases of sCJD (ACTA Neuropathol 96:116-122 (1998)), also suggest that interactions between these proteins could contribute to disease pathogenesis. Laboratory models of prion infection therefore represent a way of studying prion and prion-like mechanisms of disease that can be applied to other neurodegenerative diseases triggered by misfolded proteins. We are interested in understanding the molecular mechanisms underlying PrP amyloid formation and have begun to approach this issue using both in vitro and in vivo model systems. This project focuses primarily on 1) understanding the pathways of PrP amyloid formation and spread, 2) understanding how protein aggregation and disaggregation are controlled by the cell and, 3) studying how mutations and amino acid polymorphisms in PrP influence PrPSc amyloid formation in human forms of prion disease. Since PrPSc formation and spread appear to be mechanistically similar to the formation and spread of amyloid in other neurodegenerative diseases, the results of our prion studies will likely be broadly applicable to other diseases of protein misfolding and deposition. The ordered aggregation of PrPSc, A, or other amyloid proteins during neurodegeneration is thought to be critical to the pathogenesis of neurodegenerative protein misfolding diseases such as prion disease and AD. However, the processes by which these aggregates form and the mechanisms by which the cell can degrade them remains poorly understood. In earlier studies of how prions interact with cells, we showed that the uptake and disaggregation of prions varied by strain (J. Virol. 87: 11552-61 (2013), Annual Report 2013; Am. J. Pathol. 184: 3299-3307 (2014), Annual Report 2014) suggesting that the composition of PrPSc aggregates differed between strains. In 2021, post-doctoral fellow Dr. Daniel Shoup published the results of a study looking at how cells disaggregate PrPSc from different prion strains during the initial stages of prion infection. His data demonstrated that the sizes and stabilities of PrPSc change during cellular uptake and degradation. These changes vary with the prion strain, potentially impacting the ability of a given prion strain to infect cells. In 2021, Dr. Shoup extended his work on this project and is currently studying 1) how the conformation of PrPSc taken up by the cell changes over time, 2) which cellular proteases and processes are involved in degrading PrPSc and, 3) how the properties of PrPSc newly formed by the cell change over time. In 2021, Dr. Shoup continued experiments to optimize an in vitro protein re-folding assay using purified mammalian chaperones. He established purification procedures for the various chaperones required for his assay. He also began to optimize the conditions needed to use this cell-free system to study how PrPSc aggregates from different prion strains are unfolded and refolded by cellular chaperones under physiological conditions. These studies will provide important insights into how cellular chaperones interact with PrP aggregates. In addition, since chaperones can be cell type specific, his work may also help to elucidate why some cells are highly susceptible to prion infection and others are not. In 2021, Dr. Shoup continued experiments to adapt his in vitro chaperone refolding system to study the interactions between the SARS-CoV-2 viral spike protein and its cellular receptor ACE2 in the presence and absence of cellular chaperones. Specifically, he established purification procedures for the SARS-CoV-2 viral spike protein and ACE2. These studies will help us to understand how changes in spike protein structure allow SARS-CoV-2 to enter cells and provide an easily manipulatable in vitro system to identify inhibitors of the spike protein/ACE2 interaction. Progress on both protein refolding projects was significantly impeded starting in the second quarter of 2021 due to COVID-19 related supply shortages in the low protein binding tubes that are essential for these assays. However, Dr. Shoup is looking for alternate tubes to use and we are hopeful that this project can resume before the end of the year. We have an ongoing collaboration with Dr. Pedro Piccardo using mass spectrometry (MS) to study BSE-infected non-human primates (NHP). These animals develop a neurodegenerative disease characterized by accumulation of PrPSc, hyper-phosphorylated tau, and alpha synuclein (J Gen Virol 95:1612-16-18 (2014)) in some brain regions but not others. We have used MS to do a proteomics study to try to determine the potential molecular mechanisms underlying the different disease pathogenesis observed in two different regions of the brain. Statistical analysis of the proteomics data revealed that some of the data sets were less robust than others, an issue directly related to the intractable problems associated with the Agilent 6550 iFunnel Q-TOF mass spectrometer in our lab. In 2020, we were unable to move forward with this project due to the problems with our mass spectrometer and the fact that we cannot send prion-contaminated samples to outside entities for analysis by mass spectrometry. In 2021, a new Orbitrap mass spectrometer was installed at RML which will enable us to do a more accurate quantitation of these samples and identify differences in protein expression that will then be confirmed using non-MS based techniques. This experimental model will enable us to better understand the molecular mechanisms behind neurodegeneration in complex proteinopathies.