Investigators in Charles Weissmann’s lab at the Scripps Research Institute in Jupiter, Fla., have developed a cell-based assay that showed that a cloned murine neuroblastoma cell line called N2a-PK1 responded heterogeneously to being infected with RML and 22L prions, which causes fatal neurological disease such as scrapie in sheep and bovine spongiform encephalopathy in cattle.
Working with colleagues at the Institute of Neuropathology at the University Hospital of Zurich, Switzerland, the researchers also found that, to their surprise, sibling subclones showed very different susceptibilities to the two strains, a finding that indicates that responses to prion infection can vary independently among clones.
The investigators demonstrated that four cell lines — N2a-PK1, N2a-R33, LD9, and CAD5 — had widely varying responses to the RML, 22L, 301C, and Me7 prion strains. Also, because it is cell-based, the assay enabled the team to arrive at its results significantly faster than the traditional method, which infects mice and waits for symptoms to arise.
Their work was published online on Dec. 11 in the Proceedings of the National Academy of Science.
Sukhvir Mahal, a senior staff scientist in Weissmann’s lab, recently spoke with CBA News about the standard scrapie cell assay that Weissmann’s lab developed and the next phase of the project.
Can you give me some background of your work?
At Scripps Florida we are looking at how different cell lines can discriminate between particular strains of prions. The strains may originate from different species such as cattle with bovine spongiform encephalopathy or from multiple isolates of the same species as in sheep scrapie. All have been propagated in mice for several passages.
Traditionally, the only way to discriminate between the strains was to inoculate the brain homogenates into mice. The incubation period is normally between 150 and 250 days, and the length is a characteristic of the particular strain. As soon as the mice show clinical symptoms they are sacrificed and their brains removed for histological analysis.
Infection with the different strains of prions produces histological changes in different brain regions. The reason for this may be that the strains are targeting different cell types within the brain.
Charles Weissman’s lab moved to Scripps Florida in 2004 from the Prion Unit at the Medical Research Council in London. Weismann and his team in London had developed the standard scrapie cell assay where one prion strain, RML, can be propagated and passed onto neighboring cells in culture in a mouse neuroblastoma cell line called N2a. This technique allowed the study of prion infection in tissue culture rather than by the more laborious, time-consuming, and expensive method of inoculating a number of mice.
Using antibodies against the prion protein, we were able to look at how many cells were infected and the kinetics of infection. When we inoculated the infected cells back into mouse brains, the mice got sick. So we knew we were dealing with infection here, and that the cells had actually taken up infection and were able to propagate it.
It actually started with one strain and one cell line back in London. When we moved to Florida, we decided to expand on our work and look at several different strains of prions and a number of different cell lines.
Three of the cell lines we are using in our lab are from the central nervous system and the fourth is a fibroblast line. People have infected cell lines before and we know that they can propagate prion infectivity. What surprised us was the degrees [to which] the cell lines [responded] to the four strains. If you take one strain and put it on the four different cell lines, some of the cell lines may be susceptible to the strain, take up the infection, and propagate it, while others may not.
You expected each cell line to become infected?
We anticipated different responses on the cell lines from different strains. All the strains we are using are able to produce a response in vivo because in the brain we are dealing with the entire organ, which has neurons, astrocytes, and other different cell types.
In tissue culture, we are dealing with a particular cell line, which is usually derived from a clone. So it is not surprising that the cells in tissue culture are not infected by every prion strain.
We worked with four different cell lines and each required several steps of careful cloning. A low percentage of cells in the parent population may be able to propagate prion infectivity, but by repeated rounds of cloning we could obtain clones with an even better response.
We are enriching for the highest-susceptibility clones in every round of cloning, and we are picking siblings from it. There are just a small number of cells that are susceptible to prion infection, so when you isolate those cells some cells within this population may now be resistant and others may be susceptible.
We found that among siblings of the clones, susceptibility to different prion strains can vary quite a lot. So just because the parent of that clone was susceptible, it does not mean that all of the cells in that cell line will be susceptible; some of them will be resistant. We do not know why there is such heterogeneity in response.
What is the next step in this work?
Using this cell-culture technique, the cell panel assay with four cell lines and four prion strains [was] able to detect the different strains in tissue culture in under two weeks, instead of 150 to 250 days, because we did not have to wait for the mice to get sick.
Looking at strains in cell culture, the main question we had was, ‘How can cell lines discriminate between different prion strains?’ There may be cell-surface factors, receptors, or other proteins that allow a cell to propagate infectivity of one prion strain but not another. So the main question is, ‘Why are these cell lines responding in different ways?’
What do you plan to do next?
One of our plans is to determine if the strains somehow change once they have been passaged in cell lines. In other words, does the strain breed true? Does it maintain its characteristics?
We are now defining characteristics of these prion strains by putting them on our cell panel. After infection with a particular strain, cells are lysed, homogenized, and put back onto the cell panel to see if the strain responds in the same way as it did to the original prion strain from the mouse, or if it responds differently because it has now gone through a different host-a cloned cell line. Does the strain change when it has gone through one cell line versus another? Does it remain the same as it was originally in the mice brain?