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 have shown that amyloid formed from amyloid beta (Abeta) 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 Abeta may also be transmissible, infectious prions. Co-deposition of misfolded proteins during neurodegeneration, such as the co-localization of PrPSc and Abeta 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 and, 2) understanding how protein aggregation and disaggregation are controlled by the cell. 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 change during cellular uptake and degradation and 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 2022, Dr. Shoup continued his work on this project. His new data show that the conformations of both amyloid and non-amyloid forms of PrPSc differ but that they are similarly altered upon initial uptake and degradation by the cell. His data also suggest that these structural changes occur in different cellular microenvironments. He is currently writing a manuscript based on these results which should be submitted before the end of the year. In 2022, Dr. Shoup continued experiments developing an in vitro protein re-folding assay using purified mammalian chaperones. He successfully purified large amounts of several different chaperone and co-chaperone proteins. He has also purified PrPSc from two different prion strains, one that forms amyloid and one that does not. Dr. Shoup is now optimizing the conditions for using one of these chaperones in a protein folding/re-folding assay. His initial experiments have shown that the chaperone can unfold non-amyloid PrPSc in vitro. He will continue to use his 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 which chaperones can interact with PrPSc as well as how that interaction leads to disassembly and degradation of PrPSc aggregates.