Molecular Test for TSE Prions – TSEs

by Vincent Racaniello, PhD

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    00:00 This one can be done with either urine, recently it was found that prions actually excreted in human urine and we didn’t know that before, but in its original incarnation, the test was developed using nasal brushings. So it’s not the greatest test, because you have to put someone to sleep to do this, you lie them down and you put a long brush on the end of a long rod through their nose, all the way up to their olfactory neural epithelium, that's where your smell receptors are, it's right next to the brain case. So you have to put this thing all the way up there, and of course if you are awake, you are going to sneeze and they can't do it, so they have to put you to sleep. They brush it and get a little bit of the cells, and we know that prions can come out, you know your olfactory receptors, they are up there in your sinus essentially, and they are hardwired through your skull into your brain so the prions can come out and you get some of them if you have a prion disease. Then you do an ingenious test, really, really cool. It’s based sort of in PCR.

    01:01 Go back and listen to one of our introductory lectures I believe it's microbiology where I explained polymerase chain reaction, where you can amplify really, really small quantities of DNA. Here we can amplify small quantities of prion proteins. We take a little bit of these nasal brushings. If prion, pathogenic prions are present, PrPsc, they will be present in an oligomeric form. So look in that diagram at the bottom, there is what is called PrPsc oligomeric seed, so the SC versions will make aggregates coming out of your nose and then you add to that some PrPc that you've made by recombinant DNA technology and then you incubate them. And if there's PrPsc there, it’s going to convert the PrPc to more PrPsc, and these aggregates are going to grow. And they keep growing, you incubate them, this is many, many hours of incubation, they get longer and longer. And then at a certain point you stop the reaction, you sonicate it. You give it high frequency sound and you break up these aggregates, why? So that you can add more PrPc and repeat the cycle and you amplify it this way. You start out with a little bit of PrPsc and then you can do many, many cycles like 40 or 50 cycles and eventually you get tons of PrPsc and you can easily detect it by that Western blot that I showed you. So this works pretty well. It has pretty high sensitivity. Now if you show up and you have dementia and cerebellar ataxia and you're acting weird, they will take some your urine and do this test on it and see if you have a TSE or not. It's a very, very exciting development.

    02:44 Alright so at some point in the future we will be able to diagnose prion diseases, we will be able to treat them. In the meantime, let me tell you a little bit about the species barrier and why this scares us as well. I told you before you can inject animals with prion proteins and give them disease. So for example, if you take hamster PrPsc and you inject it into a hamster; they will develop a prion disease. Same species, no problem getting disease. If you take hamster PrPsc and inject it into a mouse, no disease.

    03:16 Doesn’t work very well cross species. However, if you make a mouse that is transgenic for the hamster prion, it will work. So the moral of the story is, the sequences of the PrPsc in the host and in the donor have to be what we call isologous, same protein or same species.

    03:39 If you simply give mice, a hamster PrP gene or whatever gene for whatever species you want to inoculate them with, they will get the infection. So, there is a species barrier to transmission, which is good. Which seems at first glance that this means that the thing, the prion protein, doesn't go easily between species. Well you know with every rule you make in biology, it can be broken. That's the way things are and this one is broken too. And we know already and you probably should preempt this, that the BSE PrPsc has a broad host range, if I eat meat contaminated with bovine prions, I can get a TSE. So what happened to the host range? Well there are always exceptions. Clearly some prions overcome the influence of the primary sequence of the protein on the host range and that's scary.

    04:28 That's why we are worried about BSE. We know that cow prions can affect people, obviously cow prions have a broad host range. The other prions we are worried about are those in cervids.

    About the Lecture

    The lecture Molecular Test for TSE Prions – TSEs by Vincent Racaniello, PhD is from the course Prions.

    Included Quiz Questions

    1. Isolation from a urine sample
    2. Isolation from a fecal sample
    3. Isolation from the vitreous humor
    4. Fine needle aspiration of muscle tissue
    5. Incisional biopsy of the brain
    1. Olfactory neural epithelium
    2. Cranial nuclei of midbrain
    3. Cranial nuclei of pons
    4. Central sulcus of brain
    5. Optic olfactory tracts
    1. ...isologous.
    2. ...linked by hydrogen bonding.
    3. ...analogous.
    4. ...autologous.
    5. ...heterologous.

    Author of lecture Molecular Test for TSE Prions – TSEs

     Vincent Racaniello, PhD

    Vincent Racaniello, PhD

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    Really Gr8!
    By Joel C. on 26. February 2023 for Molecular Test for TSE Prions – TSEs

    really gr8. to the point. fascinating. this whole program is quality cme. _JC