---start--- q: describe 3 pieces of evidence that indicate PrP is involved in TSEs and also describe a model that can explain the proliferation of an infectious particle containing no nucleic acid. Dr Pehrson - well known for his statement that "lipids are our friends" :) Today, Dr Pehrson will discuss prion diseases - transmissible spongiform encephalopathies. slide: what these do to your brain. see all those holes? the vacuoles are what caused the diseases to be called "spongiform." You do not need to be a neurologist to know it is not good to have holes all over your brain. This is a group of related diseases, probably until recently most well known was scrapie, seen in sheep - but also these diseases are seen in people and more recently in people; in other animals. Features of TSEs 1. spongiform degeneration of brain 2. dementia, ataxia, other neuro signs. dz is progressive and untreatable; leads to death 3. all forms are transmissible; also there are other genetic and spontaneous forms! also they are not infectious animal to animal - mode involves consumption in general or via transplants and such. not from shaking hands. 4. often prolonged incubation times (time from exposure to onset of clinical signs), years to decades 5. all caused by related unconventional infectious agents. the prion. It is the nature of this infectious agent which calls our attention to this disorder (family of these disorders) because it is so very different from others. Two nobel prizes have been granted to people studying these suckers. naturally occuring TSEs: man: Creutzfeldt-Jakob dz, Gerstmann straussler scheinker syndrome, Kuru, fatal familial insomnia sheep/goats: scrapie (b/c animals lean on fence, and scrape themselves) cattle: BSE cats: FSE mink: transmissible mink encephalopathy (TME) ungulates: spongiform encephalopathies of nyala, gemsbok, orynx, kudu, rocky mountain elk, captive mule deer. these may be related to the BSE form. so, human diseases: CJD - usually this dz occurs spontaneously and very rarely. this is the most common TSE in people and occurs about 1:1000000. signs of dementia progress rapidly after onset. GSSS is typically genetic or spontaneous - pattern of brain degeneration causes prominent ataxia - incidence more like 1 in 10 million. Kuru is a subform of disease found in a group of aborigines who ate their ancestors. not a good idea. they ate brains of dead relatives to honor them or pass knowledge or something. they thought it was a good idea. but, the disease was spread this way. fatal familial insomnnia - more recently found, related to CJD but has different pattern of brain degeneration and causes chronic insomnia leading to death. the effects of these diseases are appalling, but they are rare diseases. sheep/goats: scrapie - unusal thing about scrapie is the transmission from animal to animal which isn't seen with any other forms of TSE. but there is sort of a chronic, endemic level of this among sheep in many parts of the world. perhaps tick vector? Dr Pehrson does not think so. cow: BSE: mad cow disease - appeared in late 80s - very dramatically and became big problem in britain. up to now, has probably killed 150,000 cattle in a year. indirectly led to more deaths b/c government forced farmers to kill their cows in order to restore public faith in the industry. controversy - can this disease be spread to humans?? there has been a disease called variant CJD - aka vCJD which strongly correlates with BSE prion. So we think BSE can occur in people and has occured in people. But due to prolonged incubation it's hard to make the connection. the BSE, by the way, probably started as scrapie - cows were being fed ruminant derived diets and they changed the rendering process like Dr Smith explained. this allowed the infectious agent to pass through into the cows who ate the stuff. then of course they were also feeding cattle to cattle so after a cow got infected (so, cow zero probably got it from sheep...) it spread to more cows and so forth mink TME: similar thing with BSE occured with TME in 60s. once food source was removed, problem went away. it wasn't spread vertically among mink. cat diseases, kudu, etc - all probably downstream from cow, due to cow stuff being fed to other animals. properties of the infectious agent involved in scrapie and related diseases: paper by Stan Prusiner, Science 216, 136 (1982) stable at 90 C for 30 min low molecular weight particles (minimum 50,000 daltons or less - size of protein) hydrophobic protein resistant to ribonucleases and deoxyribonucleases resistant to UV irradiation at 254 nm resistant to psoralen photoadduct formation resistant to Zn++ catalyzed hydrolysis resistant to NH2OH chemical modification (those are txs which should destroy ribonucleases) so Prusiner called it a "prion" for want of a better word which you can say "pry-on" or "pree on". this is a small proteinaceous infectious particle resistant to procedures used to destroy nucleic acids. all other infectious particles contain nucleic acid. what is going on? is this an infectious protein? the concept of an infectious protein was proposed as a theory back in the 60s. How could a protein create transmissible disease? ** what kind of mechanism could lead to a protein having property of transmissible infectious agent? how does this explain the properties of prion diseases? here is one suggestion: the idea is not that proteins replicate themselves. nucleic acids can, proteins can't. this is a recruitment model - an infectious form of protein can recruit new protein to become infectious. the protein has two distinctly different states. evidence in terms of prion dzs is that the nature of the states are conformational differences- normal shape/conformation of protein is in animal, no problem, it's doing its regular job. BUT for some unknown reason, there is an alternative conformation - the prion state -which creates disease. see diagram in handout: prion protein cellular form <====> short lived monomer prion protein infectious polymer made of multiple short lived monomers so when this form gets into cell, the short lived monomers glom onto it - it grows and grows - basically this infectious form is a seed or template - it stabilizes the short lived monomer. the short lived monomer in the absence of the infectious form is normal, will not stay in that form, will not spontaneously form the polymer (except, perhaps, in spontaneous forms of these TSEs). but when the infectious polymer gets in there, the short lived monomers start to add onto it instead of reverting to normal form. this is just one theory here. it explains how you have a constant pool of normal protein which is drawn on to create increasing amounts of abnormal protein. the abnormal form of the protein, when polymerized like this, becomes resistant to the normal processes which would make it go back to the regular form. so you accumulate this insoluble aggregate and interfere with cellular processes, kill the cell, get relaesed and then into nearby cells. this material has to come from the outside of the cell into the inside of the cell and we're not quite sure how it does that. may be on surface of the cell. what is the direct evidence for prion induced diseases? well, we do not have a smoking gun or anything and there are skeptics out there but it is clear this protein is involved. The protein is called prion protein: PrP was discovered by Prusiner 1. the normal protein, which is called PrPc, is expressed in normal and infected brains at similar levels. This is interesting [They did work with mice, injecting material into brains and stuff, and they found this stuff out b/c in rodents the diseases take less time to develop and so it's easier to study.] PrPc is also expressed in non-neural tissues. it is transported to external cell surfaces and the function is unknown. 2. The abnormal protein, PrPsc, is characterized by a high resistance to protease degradation. This is the form found in the scrapie infected brain. it accumulates into large amounts inside the cell and is not ever fuond in normal brains or brains with different types of neurodegeneration 3. PrPc and PrPsc differ only in their conformation - no primary structural differences or posttranslational modification 4. PrPsc copuriies with scrapie infectivity through extensive purification Genetic evidence that PrP is involved in prion diseases -the genetic forms of human dz: CJD and GSSS - involve mutations in the gene coding for this protein - specific amino acid substitions in the gene for PrP are associated with specific inherited forms of disease. -mice with no PrP (PrP knockouts) are resistant to scrapie -incubation time of scrapie in mice is correlated to an AA substitution in PrP -transgenic mice expressing GSS form of PrP develop spontaneous neurodegeneration. transmisison of disease from these mice has been difficult. going back to that recruitment model diagram - if you have a mouse with no PrP, it won't matter if you put in infectious prion - there is no short lived monomer form being made, you can't recruit it, there is no disease! the mouse with no PrP, by the way, seems to be normal, to the extent that you can tell (who knows, maybe it can't read anymore or something...). someone is working on making PrP knockout cows, he thinks. So, how would mutations cause disease? mutant form of PrP may not cause gross change in protein, but might allow short lived monomer form to form the polymer on its own. these diseases bythe way do appear late in life. anyway, a protein doesn't have to be mutant to be involved- normal protein is recruited by the infectious polymer form. but mutations can facilitate this to occur without the seed. table p 5 of handout - other tissues also make PrP and can be infected. table shows distribution of PrP in different organs one question now raised in England- do they have to worry about blood transfusions and stuff? not sure. No simple assay exists for this agent. Bioassay, maybe - but that is a very slow test and there is a lot of species barrier - transmitting dz b/w species is harder. at this point the only way to definitely dx is brain biopsy. so there are a lot of interesting, unresolved questions. one is what are structural differences in the normal and abnormal? alpha helix vs beta sheets. PrP is alpha helix, abnormal form is beta sheet. this is pretty dramatic conformational change. what is it for? the mice without PrP seem pretty much perfectly normal so what this PrP is for is unclear. is something other than PrP involved in this process? maybe. some virologists are sure we will find the virus that causes prion diseases one day. that's because this disease is so complex. two human dzs - CJD and FFI - both involve same mutation in prion protein gene - but FFI has an additional AA change in their PrP (this change is actually a normal variation). But people with more usual form and certain mutation get CJD, and people with normal variant plus same mutation get FFI. one causes cortical damage, one causes thalamic damage. how? why? well, the only real explanation we have that fits with protein only theory is that the protein has multiple abnormal conformations, each of which causes a distinct form of the disease. people were very resistant to this idea. but virologists trying to disprove it actually generated a lot of support for it. a few years ago someone came and talked about how to fingerprint the conformation of the protein with proteases, and he showed that the different patterns of pathology did in fact correlate with different conformations of the protein. this was some of the best evidence that vCJD is caused by the same form as BSE b/c they have the same fingerprint. so that really indicates the cow disease probably caused the human disease. another guy, a virologist skeptic, has found similar stuff- that the protein can have multiple conformations. he was able to induce the formation of the abnormal conformations in vitro by mixing it with the infectious material. the pattern of the change he induced was dependent upon source of infectious material. so each abnormal conformation tends to propagate itself. ---end---