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 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. 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 in transgenic mouse models 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 (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 potentially 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 how protein aggregation and disaggregation are controlled by the cell and, 2) understanding the pathways of PrP amyloid formation and spread. 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, Abeta, and 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 prion 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. The post-doctoral fellow in the lab, Dr. Daniel Shoup, has demonstrated that the sizes and stabilities of PrPSc aggregates change during cellular uptake and degradation. He further showed that these changes vary with the prion strain, potentially impacting the ability of a given prion strain to infect cells (Shoup and Priola, Biochemistry 60: 398-411 (2021), Annual Report 2021). In 2023, Dr. Shoup published studies showing that prion aggregates contain both protease-sensitive and resistant forms of prion protein that appear to interact in a regular manner. He used this biochemical characteristic to monitor how the cell tries to unfold and degrade prions during the initial stages of prion infection. His data suggest that the ability of a prion strain to infect a cell may correlate with its ability to protect its core structure from cell-induced structural changes. In 2023, Dr. Shoup used an in vitro protein re-folding assay using purified mammalian chaperones, which he developed, to study how different chaperones interact to unfold PrPSc under physiological relevant conditions. His results show that only some chaperones are able to interact with and alter PrPSc structure. These interactions are prion strain specific and dependent upon pH. His work suggests that only certain combinations of cellular chaperones and environments are conducive to unfolding PrPSc and provides insights into why only some prion strains can infect only certain cell types. He is currently writing the manuscript for this study which should be submitted later this year. We have discovered that prion aggregates have different sizes and stabilities that may affect its ability to infect a cell and replicate (Shoup and Priola, Biochemistry 60: 398-411 (2021), Annual Report 2021). In 2023, this discovery formed the basis of a collaboration with Dr. Byron Caughey's laboratory to characterize protein aggregates found in different human neurodegenerative diseases caused by the same misfolded protein. The goal of this study is to determine if these aggregates differ depending upon the type of disease, and whether these differences impact the ability of the aggregates to induce their own formation. These studies will provide mechanistic insights into how the same misfolded protein can cause different forms of neurodegenerative disease. Finally, in 2023 Dr. Shoup initiated a pilot study to look at how regulation of chaperone expression may impact the interaction of PrP with A. If successful, his work will significantly expand our understanding of any functional relationship between PrP and A and how that may impact the progression of neurodegenerative diseases.