Research Article: Continuous Production of Prions after Infectious Particles Are Eliminated: Implications for Alzheimer’s Disease

Date Published: April 11, 2012

Publisher: Public Library of Science

Author(s): Kohtaro Miyazawa, Terry Kipkorir, Sarah Tittman, Laura Manuelidis, Jean-Luc E. P. H. Darlix. http://doi.org/10.1371/journal.pone.0035471

Abstract

Rat septal cells, induced to enter a terminal differentiation-like state by temperature shift, produce prion protein (PrP) levels 7x higher than their proliferative counterparts. Host PrP accumulates on the plasma membrane, newly elaborated nanotubes, and cell-to-cell junctions, important conduits for viral spread. To find if elevated PrP increased susceptibility to FU-CJD infection, we determined agent titers under both proliferating and arresting conditions. A short 5 day arrest and a prolonged 140 day arrest increased infectivity by 5x and 122x (>2 logs) respectively as compared to proliferating cells. Total PrP rapidly increased 7x and was even more elevated in proliferating cells that escaped chronic arrest conditions. Amyloid generating PrP (PrP-res), the “infectious prion” form, present at ∼100,000 copies per infectious particle, also increased proportionately by 140 days. However, when these highly infectious cells were switched back to proliferative conditions for 60 days, abundant PrP-res continued to be generated even though 4 logs of titer was lost. An identical 4 log loss was found with maximal PrP and PrP-res production in parallel cells under arresting conditions. While host PrP is essential for TSE agent spread and replication, excessive production of all forms of PrP can be inappropriately perpetuated by living cells, even after the initiating infectious agent is eliminated. Host PrP changes can start as a protective innate immune response that ultimately escapes control. A subset of other neurodegenerative and amyloid diseases, including non-transmissible AD, may be initiated by environmental infectious agents that are no longer present.

Partial Text

It is often stated that the normal host prion protein (PrP) converts itself into an infectious, protease resistant form (PrP-res) that causes diverse transmissible encephalopathies (TSEs). TSEs include human Creutzfeldt-Jakob Disease (CJD) and kuru, sheep scrapie, and epidemic Bovine Spongiform Encephalopathy (BSE). The recent outbreak of a virulent strain of epidemic BSE has now been largely eradicated with the removal of infected meat and livestock. While the infectious protein or prion concept has garnered a large following, i) geographically specific environmental origins of different TSEs [1], [2], [3], ii) the classic viral-like spread of these agents through the lymphoreticular system, white blood cells and dendritic cells to the brain [4], [5], iii) early innate immune responses of the host that signify host recognition of a foreign infectious agent [6], iv) abundant agent replication before PrP-res becomes detectable [7], [8], and v) the lack of any consistent strain-specific forms of “infectious” PrP [1], [9] all pose fundamental problems for the prion hypothesis. For simplicity, we here consider prions as their originally defined PrPsc form (identical to proteinase K resistant PrP-res) [10].

If host PrP is a required receptor for TSE agents, then increasing the level of PrP should increase both susceptibility to infection and the infectious titer. In Tga20 mice with 8x the levels of PrP, the incubation time of CJD and scrapie agents is reduced as compared to wt mice with 1x PrP [1], [3], [9], [30]. This shows PrP enhances susceptibility to TSE infectious agents. On the other hand, the final brain titer in Tga20 mice is not higher than in wt mice [30]. Rat SEP cell cultures that will produce high PrP levels under arresting conditions (37.5°C-2% serum) offered another opportunity to explore the relationship of titer to PrP and PrP-res in a highly controlled biological system. Uninfected SEP cells rapidly transition into a stationary non-dividing state that resembles terminal neural differentiation, and they concomitantly express ∼7x levels of PrP. When uninfected control SEP cells are maintained long term at 37.5°C-2% serum they continue to produce 5–8x levels of PrP, and when switched to standard proliferative conditions (33°C-10% serum) they immediately revert to 1x PrP levels [22].

FU-CJD infection of living SEP cells provides insight into innate host cell responses that have been difficult to sort out in animal models with complex and multiple cell type reactive changes. The cell biology of infection and control are not operative and cannot be analyzed in test tube reconstitution experiments such as PMCA. The production of extremely high levels of FU-CJD infectivity in SEP cells with ∼10x normal levels of PrP was unexpected because Tga20 mice with comparably high PrP levels have not shown increased titers compared to wt mice with 1x PrP; this is true for diverse agents including Chandler (RML) scrapie, FU-CJD, and kuru agents in Tga20 mice [3], [23], [30]. On the other hand, both SEP cells and Tga20 mice show high PrP facilitates infection. In the case of Tga20 mice, this is reflected by the more rapid progression to terminal disease, with relatively short incubation times as compared to wt mice. Similarly, the short 5 day SEP cell arrest with elevation of PrP led to an increase of the FU-CJD infectious agent close to the predicted level based on the FU-CJD doubling time. This data is in keeping with PrP as a receptor that binds to and facilitates the entry and reproduction of the TSE infectious particle. In mice, high levels of infectious particles will produce clinical signs of disease, including host astrocytic and microglial responses that can be destructive and lead to death [8], [32]. Even higher levels of agent did not kill the SEP cells here, possibly because they are not subject to destructive cellular elements. SEP cells maintained for 140 days under arresting conditions continued to produce infectious particles and high PrP levels even though they escaped arresting conditions and proliferated at the same rate as their untreated counterparts. Remarkably, titers of these proliferating FU-CJD infected, high PrP SEP cells were ∼2 logs greater than found in RML scrapie brain, i.e., 9.3 versus 7.3 logs per gram (e9 cells). This difference may, at least in part, be due to host responses to the infectious agent before PrP-res is detectable. During the initial phase of in vivo infection, non-PrP host responses clearly recognize the infectious agent [6]. Moreover, agent doubling time studies show that it can take as much as 22x longer to accumulate the same number of infectious particles in a mouse brain as compared to cultured monotypic cells [23]. This emphasizes host recognition of and defense against an invading foreign TSE agent.

Source:

http://doi.org/10.1371/journal.pone.0035471