Prion biology is a big tent. It encompasses basic biology as well as therapeutic science, human prions as well as yeast and animal prions, and increasingly, prion diseases as well as other neurodegenerative diseases, like Alzheimer’s Disease and ALS, that seem to share common mechanisms. The annual prion meeting tends to draw from topics across this spectrum, and this year’s Prion 2015 meeting, held at Colorado State University, was no exception.
About 500 prion researchers gathered in Ft. Collins, Colorado from May 26-29 to share with each other the latest and greatest in prion research. If you’ve been following Eric’s blog posts at cureffi.org, then you’ve gotten a sense of the nitty gritty details. As you might expect given the conference’s location, many of the talks focused on chronic wasting disease, the prion disease that has now been endemic in deer and elk in Colorado for nearly 50 years, and has now been reported in 23 U.S. states. But if, like us, your main interest is human prion diseases – Creutzfeldt-Jakob disease, fatal familial insomnia, and Gertsmann-Straussler-Scheinker disease – and the prospects for a treatment or cure, here’s the lowdown on some of the best tidbits from the conference.
The Prusiner lab at UCSF offered two exciting updates on its drug discovery program. For background, recall that prions come in different strains. At the atomic level, these represent different shapes of misfolded protein, and at the level of the patient, that translates into slightly different types of disease, with, for instance, some differing symptoms or a longer or shorter disease course. Back in 2013, postdoctoral researcher David Berry announced the unfortunate news that IND24, a chemical compound which the Prusiner lab had spent years developing as a potential drug, had proven to be effective against some strains of prions but not others. In particular, the drug extended survival time by more than 50% in mice infected with a commonly used strain of mouse prions called RML, but it had no effect in mice infected with prions from CJD patients [Berry 2013]. What’s more, even when the drug did work, the prions gradually changed shape and developed drug resistance. This was a major setback for drug discovery efforts, and at the time, we didn’t expect to hear any more about IND24.
But to our surprise, there were two interesting updates on IND24 this year. First, Dr. Berry announced that IND24 works surprisingly well in mice infected with prions from elk dying of chronic wasting disease. While this doesn’t mean that IND24 will work against any human prions, and we shouldn’t expect it to head to clinical trials, it is a surprising and exciting breakthrough. This chemical was discovered by studying libraries of different chemicals in mouse cells infected with mouse prions, so the fact that it just happens to work against elk prions too is proof that different prion strains do have some things in common, and finding a drug to target the prion strains we care about - those that kill humans - might be more feasible than we thought. What’s more, the elk prions didn’t develop drug resistance the way that mouse prions did with IND24. Therefore, there is a chance that the “drug resistance” problem we’ve been worried about for the past few years might not be quite as bad as we thought. As of now remains to be seen whether the human prion strains that we care about will be able to develop drug resistance or not.
The second update was that Kurt Giles presented new data on the effect of different dosing regimens for IND24 in mice. Again, the compound doesn’t work against human prions, but it was interesting to speculate that the new findings for mouse prions might be our first glimpse at what a dosing regimen could one day look like for a drug against human prions. Surprisingly, it turns out that the drug is most effective when given intermittently, rather than continuously. Giving a drug intermittently isn’t totally unprecedented - for instance, many chemotherapy drugs are given intermittently, but that is simply because they are too toxic for patients to take them continuously. The finding that IND24 actually worked better when it was taken in an on/off/on/off fashion was interesting. Also, and perhaps less surprisingly, the earlier the mice were given the compound, the more effective it was. Incredibly, when mice received the compound prophylactically — before being infected with prions — they lived more than four times as long as untreated mice. That gives us hope that when we one day have a drug against human prions, it might have a really dramatic effect in genetic mutation carriers, who could start taking a drug years before symptoms develop.
Dr. Byron Caughey of the Rocky Mountain Labs in Hamilton, MT, gave one of two keynote addresses on the opening evening of the conference. A major focus of his talk was RT-QuIC, or real-time quaking induced conversion, and efforts by his lab and Ryuichiro Atarashi’s lab in Japan to develop and improve this assay. As evidenced by the number of talks and posters that made reference to RT-QuIC over the course of the conference, this assay represents a major development and area of rapid growth in the prion disease field – and it is a development that has direct relevance to patients.
So what is RT-QuIC, how does it work, and why does it matter? First, a quick refresher on the prion protein. In its healthy form, the human prion protein is found in all of us throughout life. Most of the time this small protein hangs out on the cell surface, doing its normal job. We don’t know exactly what this normal job is, but we do have an idea of what can go wrong. In prion disease, this normally healthy protein changes shape, or misfolds. Misfolded prion protein forms “prions” that initiate a domino effect by recruiting other molecules of healthy prion protein to likewise misfold. For reasons that also aren’t well understood, these prions are toxic to brain cells, so their spread across the brain results in devastating disease.
The handy thing about RT-QuIC is that it allows researchers to recapitulate the “domino effect” of prion formation in a dish. Basically, a sample of patient biofluid is added to a reaction mix, and after several hours of heating and shaking, the reaction produces a signal. The timing and strength of this signal tell you, with impressive reliability, whether or not a cascade of protein misfolding, triggered by a prion, has occurred – and from this, you can infer whether there were prions in the patient sample. While RT-QuIC has been around for several years, it continues to improve. New reaction conditions have raised the sensitivity of the assay to patient cerebrospinal fluid, which can be acquired though a lumbar puncture (spinal tap) [Orru 2015], and also to nasal brushings, which can be collected from the upper chambers of the nasal cavity [Orru 2014]. While these procedures may sound a bit invasive, they are much less invasive than taking a brain biopsy – and they serve the major goal of confident identification of prion disease patients while they are still alive. This is a critical point because one thing we know from years of drug discovery efforts is that in order to help to treat prion disease patients, we are going to have to reach them as early as possible. Improved early diagnosis is critical, not just to avoiding the diagnostic odysseys that so many families suffer through, but also to imagining the clinical trials of the future that will eventually enable life-saving drugs.
Dr. John Collinge of the MRC Prion Unit in London gave a fascinating presentation largely focused on kuru, the prion disease that afflicted the Fore people of Papua New Guinea. Kuru represents a unique chapter in the history of prion disease, as it was transmitted person-to-person through ritualistic cannibalism. This practice was ended by about 1961, leading to the tapering of the epidemic.
Dr. Collinge devoted a good part of his talk to narrating the history of kuru. In the process he showed photos and videos from Papua New Guinea, displaying the lush landscape as well as a number of interviews with the Fore people. He shared some striking photos of the field research station that MRC Prion Unit set up in the mountains of the Fore region, including thatched walls, a hand-powered centrifuge, and a solar-powered freezer. It was humbling to imagine the amount of effort and ingenuity it took to do biomedical research in the middle of the rainforest.
From this history, Dr. Collinge proceeded to share data gathered from the MRC Prion Unit’s continuing work with the Fore. Although the kuru epidemic was already subsiding by the mid-1960s, Dr. Collinge discussed the wide variation in kuru incubation time. While the average time between exposure to kuru and development of disease is thought to have been around 12 years, a handful of people exposed to kuru prions in the 1960s presented with disease in the 1990s, and an even more recent case was identified just last year. Dr. Collinge also shared a remarkable finding regarding survivors of the kuru epidemic. His group had previously observed that some Fore people exposed to kuru seemed to be protected by a variant in their PRNP gene [Mead 2009]. Specifically, in these individuals, a change in their PRNP genetic sequence led to production of the amino acid valine (V) instead of the usual glycine (G) at position 127. To further investigate this effect, the MRC prion unit has now made mice that produce human PrP with this “G127V” protective variant. They attempted to infect these mice with a number of human prion strains including kuru and sporadic CJD, and found mice with the protective variant were less susceptible to disease. In fact, mice with two copies of the G127V variant appeared to be completely protected against infection with prions [Asante 2015].
This story is hot off the press, and it’s worth taking a moment to reflect on what it does and doesn’t mean. Unfortunately, for those of us at risk for prion disease, there are no immediate implications in terms of our own risk. The G127V variant happens to have been present in the Fore people, and because it provided such a strong survival advantage during the kuru epidemic, its prevalence has risen among the Fore. But according to Dr. Collinge, this variant has not been seen anywhere else in the world. So, this is not exactly Christmas come early in terms of a protective variant hiding out in our own genomes. However, that’s not to say that in the longer term, this fascinating discovery may not have therapeutic implications. It will take a lot of time and a lot of work, but understanding how the G127V variant changes the prion protein, and how exactly this makes the protein resistant to conversion into the disease form, could help us to design therapeutic strategies that mimic this protective effect. It will be interesting to follow this story over time, and to see what this example of natural resistance may be able to teach us in the fight against prions.
Finally, a quick reflection on the structural biology of prions. A number of talks at the conference, such as those by Dr. Holger Wille and Dr. Jesús Requena, and a good part of Dr. Byron Caughey’s keynote – were focused on the molecular structure of prions. The question being asked here is basically: we know that prions are the bad actors are prion disease, but what exactly do they look like, on the atomic level? While this may seem like a nitty-gritty topic that is pretty removed from therapeutics, in some ways it is very relevant to drug development and it’s exciting to see that this part of the field is very active.
In short, even though we don’t know the normal job of the prion protein, we do have a reasonable sense of what the prion protein looks like in this healthy state, thanks to a suite of techniques that allow us to indirectly “visualize” these tiny molecules by gathering indirect information on their structure. We also know that a change in shape occurs when prions go wrong. But we don’t know exactly what the misfolded prions look like, in part because once misfolded, prions tend to clump up, making them unsuited for many traditional structural biology techniques.
There are all sorts of open questions here. It is thought that prions contain more than one misfolded prion protein, but the jury is still out on how many. Likewise, we’re not sure yet whether other materials besides misfolded prion protein are bound up in the infectious particles. Different prion strains appear to differ in conformation, but how different are they? A high-resolution picture of prion structure could help to shed light on how prions replicate; on the similarities and differences between different prion strains; on why, as in the IND24 example discussed earlier, drug resistance emerges under some circumstances but not others; and on why, as with the G127V variant, certain genetic changes seem to confer resistance against prion conversion. And all of these insights could potentially help us to design and discover prion-targeting drugs in smarter, more directed ways.
In summary, it was an exciting few days. It is always heartening to be reminded of the work going on all over the globe to better understand prion biology and prion disease. Special thanks to those researchers who were willing to share unpublished work, giving us a glimpse at where the field is headed in the near future.