Tag Archives: Laura Manuelidis

A possible new player

Drug development is very hard because we don’t know all the players inside the cell. A recent paper describes an entirely new class of player — circular DNA derived from an ancient virus.  The authoress is Laura Manuelidis, who would have been a med school classmate had I chosen to go to Yale med instead of Penn.   She is the last scientist standing who doesn’t believe Prusiner’s prion hypothesis.  She didn’t marry the boss’s daughter being female, so she married the boss instead;  Elias Manuelidis a Yale neuropathologist who would be 99 today had he not passed away at 72 in 1992.

The circular DNAs go by the name of SPHINX  an acronym  for  Slow Progressive Hidden INfections of X origin.  They have no sequences in common with bacterial or eukaryotic DNA, but there some homology to a virus infecting Acinebacter, a wound pathogen common in soil and water.

How did she find them?  By doggedly pursuing the idea the neurodegenerative diseases such as Cruetzfeldt Jakob Disease (CJD) and scrapie were due to an infectious agent triggering aggregation of the prion protein.

As she says:  “The cytoplasm of CJD and scrapie-infected cells, but not control cells, also contains virus-like particle arrays and because we were able to isolate these nuclease-protected particles with quantitative recovery of infectivity, but with little or no detectable PrP (Prion Protein), we began to analyze protected nucleic acids. Using Φ29 rolling circle amplification, several circular DNA sequences of <5 kb (kilobases) with ORFs (Open Reading Frames) were thereby discovered in brain and cultured neuronal cell lines. These circular DNA sequences were named SPHINX elements for their initial association with slow progressive hidden infections of X origin."

SPHINX itself codes for a 324 amino acid protein, which is found in human brain, concentrated in synaptic boutons.  Strangely, even though the DNAs are presumably viral derived, they contain intervening sequences which don't code for protein.

The use of rolling circle amplification is quite clever, as it will copy only circular DNA.

Stanley Prusiner is sure to weigh in.  Remarkably, Prusiner was at Penn Med when I was and was even in my med school fraternity (Nu Sigma Nu)  primarily a place to eat lunch and dinner.  I probably ate with him, but have no recollection of him whatsoever.

Circular DNAs outside chromosomes are called plasmids. Bacteria are full of them. The best known eukaryote containing plasmids is yeast. Perhaps we have them as well. Manuelidis may be the first person to look.

Kuru continues to inform

Neurologists of my generation were fascinated with Kuru, a disease of the (formerly) obscure Fore tribe of New Guinea. Who would have thought they would tell us a good deal about protein structure and dynamics?

It is a fascinating story including a Nobelist pedophile (Carleton Gajdusek) https://en.wikipedia.org/wiki/Daniel_Carleton_Gajdusek and another (future) Nobelist who I probably ate lunch with when we were both medical students in the same Medical Fraternity but don’t remember –https://en.wikipedia.org/wiki/Stanley_B._Prusiner

Kuru is a horrible neurodegeneration starting with incoordination, followed by dementia and death in a vegetative state in 4 months to 2 years. For the cognoscenti — the pathology is neuronal loss, astrocytosis, microglial proliferation, loss of myelinated fibers and the kuru plaque.

It is estimated that it killed 3,000 members of the 30,000 member tribe. The mode of transmission turned out to be ritual cannibalism (flesh of the dead was eaten by the living before burial). Once that stopped the disease disappeared.

It is a prion disease, e.g. a disease due to a protein (called PrP) we all have but in an abnormal conformation (called PrpSc). Like Vonnegut’s Ice-9 (https://en.wikipedia.org/wiki/Ice-nine) PrPSc causes normal PrP to assume its conformation, causing it to aggregate and form an insoluble mess. We still don’t know the structure of PrPSc (because it’s an insoluble mess). Even now, “the detailed structure of PrPSc remains unresolved” but ‘it seems to be’ very similar to amyloid [ Nature vol. 512 pp. 32 – 34 ’14]. Not only that, but we don’t know what PrP actually does, and mice with no PrP at all are normal [ Nature vol. 365 p. 386 ’93 ]. For much more on prions please see https://luysii.wordpress.com/2014/03/30/a-primer-on-prions/

Prusiner’s idea that prion diseases were due to a protein, with no DNA or RNA involved met with incredible resistance for several reasons. This was the era of DNA makes RNA makes protein, and Prisoner was asking us to believe that a protein could essentially reproduce without any DNA or RNA. This was also the era in which X-ray crystallography was showing us ‘the’ structure of proteins, and it was hard to accept that there could be more than one.

There are several other prion diseases of humans (all horrible) — mad cow disease, Jakob Creutzfeldt disease, Familial fatal insomnia, etc. etc. and others in animals. All involve the same protein PrP.

One can take brain homogenates for an infected animal, inoculate it into a normal animal and watch progressive formation of PrPSc insoluble aggregates and neurodegeneration. A huge research effort has gone into purifying these homogenates so the possibility of any DNA or RNA causing the problem is very low. There still is one hold out — Laura Manuelidis who would have been a classmate had I gone to Yale Med instead of Penn. n

Enter [ Nature vol. 522 pp. 423 – 424, 478 – 481 ’15 ] which continued to study the genetic makeup of the Fore tribe. In an excellent example of natural selection in action, a new variant of PrP appeared in the tribe. At amino acid #127, valine is substituted for glycine (G127V is how this sort of thing is notated). Don’t be confused if you’re somewhat conversant with the literature — we all have a polymorphism at amino acid #129 of the protein, which can be either methionine or valine. It is thought that people with one methionine and one valine on each gene at 129 were somewhat protected against prion disease (presumably it affects the binding between identical prion proteins required for conformational change to PrPSc.

What’s the big deal? Well, this work shows that mice with one copy of V127 are protected against kuru prions. The really impressive point is that the mice are also protected against variant Creutzfedlt disease prions. Mice with two copies of V127 are completely protected against all forms of human prion disease . So something about V/V at #127 prevents the conformation change to PrPSc. We don’t know what it is as the normal structure of the variant hasn’t been determined as yet.

This is quite exciting, and work is certain to go on to find short peptide sequences mimicking the conformation around #127 to see if they’ll also work against prion diseases.

This won’t be a huge advance for the population at large, as prion diseases, as classically known, are quite rare. Creutzfeldt disease hits 1 person out of a million each year.

There are far bigger fish to fry however. There is some evidence that the neurofibrillary tangles (tau protein) of Alzheimer’s disease and the Lewy bodies (alpha-Synuclein) of Parkinsonism, spread cell to cell by a ‘prionlike’ mechanism [ Nature vol.485 pp. 651 – 655 ’12, Neuron vol. 73 pp. 1204 – 1215 ’12 ]. Could this sort of thing be blocked by a small amino acid change in one of them (or better a small drug like peptide?).

Stay tuned.

The prions within us

Head for the hills. All of us have prions within us sayeth [ Cell vol. 156 pp. 1127 – 1129, 1193 – 1206, 1206 – 1222 ’14 ]. They are part of the innate immune system and help us fight infection. But aren’t all sorts of horrible disease (Bovine Spongiform Encephalopathy aka BSE, Jakob Creutzfeldt disease aka JC disease, Familial Fatal Insomnia etc. etc.) due to prions? Yes they are.

If you’re a bit shaky on just what a prion is see the previous post which should get you up to speed — https://luysii.wordpress.com/2014/03/30/a-primer-on-prions/.

Initially there was an enormous amount of contention when Stanley Prusiner proposed that Jakob Creutzfeldt disease was due to a protein forming an unusual conformation, which made other copies of the same protein adopt it. It was heredity without DNA or RNA (although this was hotly contended at the time), but the evidence accumulating over the years has convinced pretty much everyone except Laura Manuelidis (about whom more later). It convinced the Nobel Prize committee at any rate.

JC disease is a rapidly progressive dementia which kills people within a year. Fortunately rare (attack rate 1 per million per year) it is due to misfolded protein called PrP (unfortunately initially called ‘the’ prion protein although we now know of many more). Trust me, the few cases I saw over the years were horrible. Despite decades of study, we have no idea what PrP does, and mice totally lacking a functional Prp gene are normal. It is found on the surface of neurons. Bovine Spongiform Encephalopathy was a real scare for a time, because it was feared that you could get it from eating meat from a cow which had it. Fortunately there have been under 200 cases, and none recently.

If you cut your teeth on the immune system being made of antibodies and white cells and little else, you’re seriously out of date. The innate immune system is really the front line against infection by viruses and bacteria, long before antibodies against them can be made. There are all sorts of receptors inside and outside the cell for chemicals found in bacteria and viruses but not in us. Once the receptors have found something suspicious inside the cell, a large protein aggregate forms which activates an enzyme called caspase1 which cleaves the precursor of a protein called interleukin 1Beta, which is then released from some immune cells (no one ever thought the immune system would be simple given all that it has to do). Interleukin1beta acts on all sorts of cells to cause inflammation.

There are different types of inflammasomes and the nomenclature of their components is maddening. Two of the sensors for bacterial products (AIM, NLRP2) induce a polymerization of an inflammasome adaptor protein called ASC producing a platform for the rest of the inflammasome, which contains other proteins bound to it, along with caspase1 whose binding to the other proteins activates it. (Terrible sentence, but things really are that complicated).

ASC, like most platform proteins (scaffold proteins), is made of many different modules. One module in particular is called pyrin (because one of the cardinal signs of inflammation is fever). Here’s where it gets really interesting — the human pyrin domain in ASC can replace the prion domain of the first yeast prion to be discovered (Sup35 aka [ PSI+ ] — see the above link if you don’t know what these are) and still have it function as a prion in yeast. Even more amazing, is the fact that the yeast prion domain can functionally replace ASC modules in our inflammasomes and have them work (read the references above if you don’t believe this — I agree that it’s paradigm destroying). Evidence for human prions just doesn’t get any better than this. Fortunately, our inflammasome prions are totally unrelated to PrP which can cause such havoc with the nervous system.

Historical note: Stanley Prusiner was a year behind me at Penn Med graduating in ’67. Even worse, he was a member of my med school fraternity (which was more a place to get a decent meal than a social organization). Although I doubtless ate lunch and dinner with him before marrying in my Junior year, I have absolutely no recollection of him. I do remember our class’s medical Nobel — Mike Brown. Had I gone to Yale med instead of Penn, Laura Manuelidis would have been my classmate. Small world