Prions
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Aguzzi et al.: B lymphocytes and neuroinvasion
...comentary by Paul Brown
...commentary by Gretchen Vogel
Well worth noting: selected abstracts
Leucine rich repeats: cross-beta yes, but no disease yet
Irish nvCJD concern
Similarities between inclusion-body myositis, Alzheimer, and prion disease

Spongiform encephalopathies: B lymphocytes and neuroinvasion

Nature commentary by Paul Brown NINDS
Actual article is offline and no abstract has appeared in Medline
The latest episode in the long-running serial of scrapie pathogenesis appears on page 687 of this issue, with a further molecular dissection of genetically altered mice. The Swiss team has taken advantage of the fact that the gene encoding the prion protein PrP -- the leading candidate for the cause of all transmissible spongiform encephalopathies, including scrapie and Creutzfeldt‚Jakob disease (CJD) -- can be made inoperative (that is, 'null'), without evident harm to the mice. Working backwards from the target organ of disease (the brain) to peripheral (intraocular, intravenous or intraperitoneal) sites of infection, the authors have used surgical transplants in combination with normal and genetically altered mice in an effort to decode the pathogenesis of disease at the molecular level. They show that differentiated B lymphocytes are important for neuroinvasion -- a finding with both public-health and therapeutic implications.

Intracerebral inoculation of the scrapie agent into a null mouse does not produce disease. However, intracerebral inoculation of a null mouse harbouring a small, PrP-expressing neural transplant destroys the graft, although the surrounding tissue is free from disease. In contrast, inoculation by peripheral routes does not destroy the graft: intraocular inoculation fails despite the fact that, in normal animals, the optic nerve is an efficient route of brain infection; and intraperitoneal or intravenous inoculation do not destroy the graft, even after lethal irradiation and transplantation of PrP-expressing haematopoietic stem cells (although these mice do develop infection of the spleen).

This series of experiments builds on a foundation of studies dating back to the 1960s, when Pattison and Millson established the widespread extraneural distribution of infectivity in scrapie-affected sheep and goats. The chief architects during the 1970s were Hadlow and Ecklund, followed, in the 1980s, by Fraser and Dickinson, and by Kimberlin and Walker. The importance of their contributions cannot be overemphasized in an era in which the power of molecular biology and accelerated publication has created a misguided tendency to neglect as already dated any research completed more than a few years earlier.

Thanks to them, and subsequent researchers (Kuroda, Diringer, Kitamoto and Lasmezas), we already know a good deal about the pathogenesis of transmissible spongiform encephalopathies. After introduction of the scrapie agent into various peripheral sites, including the stomach, infectivity first appears in the lympho- reticular system ã tonsils, thymus, lymph nodes, and especially in spleen, in which a primary phase of replication occurs in B-cell-rich tissue fractions.

Infectivity in these and other widely separated body organs implies that the infectious agent travels through the bloodstream, and this has been explicitly shown by isolation of infectivity from blood during both the pre- clinical and clinical stages of disease. Only after establishment of infectivity in peripheral organs does infectivity (and PrP) become detectable in the central nervous system; first in the spinal cord, and then in the lumbar and cervical cord segments and brain stem. Finally, genetically immunodeficient mice can partially resist intraperitoneal infection, yet they recover almost full susceptibility if immunologically reconstituted by the intraperitoneal injection of normal spleen cells.

This essential schema of scrapie pathogenesis is equally valid whether the infectious agent is a peculiar little virus or (as the Swedish Academy has just authorized) the prion protein. The observation by Klein et al., that mature B lymphocytes participate in neuroinvasion, should lead to an increasingly precise appreciation of the molecular basis of scrapie pathogenesis. For example, do B lymphocytes act as blood-borne carriers of the infectious agent, or do they act while in the spleen?

Serial-infectivity measurements of blood lymphocytes and spleen during the incubation period could be compared in normal and B-cell-deficient mice. Blood infectivity could also be evaluated in splenectomized B-cell- and T-cell-deficient mice. What would happen after infection of a null mouse with PrP-expressing intracerebral and peripheral nerve implants, or intracerebral and splenic grafts? And could combinations of immunodeficient and PrP null mice strains be created?

Neuroinvasion may turn out to have a hierarchy of options. B lymphocytes contacting terminal nerve endings in the spleen could be the most efficient route; other lymphoid-tissue sites and transfer cells might be less efficient; and direct contact with nerve endings (short-circuiting the lympho-reticular system) least efficient of all. Physical transfer of the infectious agent from a lymphoid cell to a neuron might also require a ligand molecule ã the mysterious 'protein X', for example. Among the candidates are two proteins with an affinity for PrP, which have been proposed as cell-membrane PrP receptors in this month's issue of Nature Medicine.

What about the public-health and therapeutic implications of a role for B lymphocytes in peripherally acquired disease? There is a convincing link between the 'new variant' of Creutzfeldt‚Jakob disease (vCJD) and consumption of infectious tissues from cattle with bovine spongiform encephalopathy (BSE). Moreover, the infectious agents of scrapie and CJD can be isolated from blood or blood components. The involvement of B lymphocytes in neuroinvasiveness adds another reason to consider a step that will deplete white blood cells during the commercial processing of blood, to reduce the possibility of transmitting CJD through blood products.

The outlook for therapeutic benefits is more problematic. Because we have no preclinical test for infection, nor any epidemiological clues as to who is at risk of nvCJD, pre-emptive elimination of B lymphocytes is out of the question. PrP has been found in the tonsils of patients with vCJD, but this has so far been investigated only in clinically ill patients. We have no idea when ã or even whether ã PrP might appear during the incubation phase of disease. Moreover, even if PrP is present during the incubation period, we know that neuroinvasion occurs well before neurological symptoms.

We cannot imagine that entire populations would submit to tonsillar biopsies as a screening procedure for nvCJD. Thus, for the moment, we had best not push the implications of the involvement of B lymphocytes in neuroinvasion too far from the realm of basic science. When we know precisely how B lymphocytes influence neuroinvasion, it may be possible to nullify their effect without resorting to wholesale elimination strategies.

                     1. Klein, M. A. et al. Nature 390, 687‚690 (1997). 
                     2. Bueeler, H. R. et al. Nature 356, 577‚582 (1992). 
                     3. Martins, V. R. et al. Nature Med. 3, 1376‚1382 (1997). 
                     4. Rieger, R., Edenhofer, F., Lasmezas, C. I. & Weiss, S. Nature Med. 3, 1383‚1388
                     (1997). 

B Cells May Propagate Prions

Science 278, p 2050  19 December 1997,  commentary by Gretchen Vogel 
No one knows exactly what causes "mad cow disease" and related neurodegenerative conditions, such as Creutzfeldt-Jakob disease (CJD) in humans. But this uncertainty hasn't kept researchers from wondering how the agents that cause these diseases spread from the site of infection to the brain. New work by neuropathologist Adriano Aguzzi of the University of Zurich in Switzerland and his colleagues now suggests that B cells, a type of immune cell carried in the blood, play an important role in this propagation.

In this week's issue of Nature, the Aguzzi team reports that mice lacking B cells are resistant to infection with scrapie, a sheep condition similar to mad cow disease, when they are inoculated with infectious material in areas outside the brain. If B cells are necessary for the disease to propagate, the authors reason, they may also carry the infectious agent.

In Britain, where 20 young people have already died from a new variant of CJD, possibly originating in meat and other products from cattle infected with mad cow disease, the finding has sparked fears about the safety of donated blood. Experts on the diseases say that no case of CJD in humans has ever been linked to blood transfusions. But the news--which Aguzzi presented at a closed meeting in November--has prompted calls for hemophiliacs in Britain to receive blood clotting factors made by recombinant DNA technology instead of prepared from the pooled blood of many donors. It has also led to suggestions that blood banks should remove white blood cells from donors' blood.

Nailing down how the agents that cause CJD and scrapie travel through the body has been difficult because researchers don't know exactly what to look for. Many believe that the agents are misfolded proteins called prions that propagate themselves, while others think an as yet unidentified slow-acting virus is to blame. But scientists showed as long ago as the 1970s that a wide variety of immune tissues can be infective, especially tonsils, thymus, lymph nodes, and spleen, when injected into animals' brains.

To try to pin down what particular component of the immune system carries the agent, the Aguzzi team tried to infect a number of mouse strains that had been engineered to lack specific immune cells or molecules. They found that mice lacking T cells or the immune-system protein interferon were infected as easily as normal mice, but mice that had no B cells were resistant. Out of 27 such mice, none developed symptoms of scrapie after more than a year (up to 534 days), although at least four of them did show evidence of scrapie in their brains. All other mice developed scrapie within 8 months.

Other researchers do not find the results particularly surprising. They note that the tissues previously shown to be infective are rich in B cells. More recent, unpublished work by neuroscientist Paul Brown of the National Institute of Neurological Disorders and Stroke in Bethesda, Maryland, and Robert Rohwer of the Molecular Neurovirology Unit at the Veteran's Administration Medical Center in Baltimore shows that both white blood cells and plasma from infected animals can transmit disease. And at a number of meetings Rohwer has reported that he and his colleagues have infected at least one hamster--out of 22 tested--with scrapie through a transfusion.

The researchers caution, though, that the results only show that blood-borne transmission is possible in the laboratory, and say nothing about the likelihood of it in humans or animals. They point out that no human case of CJD has been traced to a blood transfusion. There is a "tremendous amount of epidemiology that all speaks against the possibility of blood-borne transmission of the agent," Aguzzi says. Rohwer points out, however, that while those results apply to classic CJD, with which "we have been living since the very first transfusion," the situation may be different for the U.K.'s new CJD variant.

There's at least one indication that the immune system could play a bigger role in transmitting the new variant. While doctors have never spotted abnormal prion proteins in the tonsils of patients with classic CJD, in new variant patients, tonsils are "full of abnormal proteins," Aguzzi says.

Aguzzi suspects that the B cells in tonsils carry the prions. But it is not clear whether all B cells harbor the infectious agent, or if only a subset do so. Says Aguzzi, "There is still a tremendous amount of work to be done." That's surely one of the only things on which all researchers in the field can agree.

Similarities between inclusion-body myositis, Alzheimer and prion-disease brain

Journal of Neuropathology and Experimental Neurology 1996; 55(N5): 631 abstract number 106
AU Askanas,V.; Engel,W.K.
IBM is the most common progressive and debilitating muscle disease in aging persons. Pathologic hallmarks of IBM are vacuolated muscle fibers (VMFs) containing paired-helical filaments (PHFs) and congophilia. Recently, we have identified abnormal accumulations within these fibers of:

a) AIzheimer specific proteins B-amyloid protein, N- and C-epitopes of beta-amyloid precursor protein, hyperphosphorylated tau, apolipoprotein E , alpha1-antichymotrypsin, ubiquitin, and transforming growth factor beta;

b) beta-APP mRNA;

c) cellular prion protein and mRNA.

The fibers are similar to Alzheimer fibers: both contain hyperphosphorylated tau (tau-P), ApoE, and UBI. Ultrastructural immunolocalization of proteins accumulated in IBM VMFs shows:

   Structure            Abeta  N-betaAPP  C-APP  UBI  Tau-P  ApoE  Prion
   PHFs                 -     -       -      +     +     +      +
   6-10 nm Filaments    +     -       -      +     -    +/-     +
   Amorphous Material   +     +       +      +     -    +/-     +
   Flocculo-membranous  +     +       +      +     -    +/-     +
Striking similarity between cellular phenotypes of IBM muscle and Alzheimer and prion-disease brain may result from a cascade of similar pathogenic steps occurring in the muscle-fiber and the neuron but provoked by different etiologies; multiple causes producing the upregulation of a common cellular gene, whose protein is a transcription factor for BAPP and/or PrPc; a tie-specific intracellular aging change that may be a requisite enhancing mechanism.

Irish nvCJD concern

Lancet 12.20.97  Karen Birchard 
The Irish Department of Health has finalised arrangements for notification and counselling of 467 people given a radiological dye manufactured from the plasma of a UK blood donor who died subsequently of new variant Creutzfeld-Jakob disease (nvCJD).

The product, Amerscan Pulmonate II (Nycomed Amersham, UK) was recalled in mid-November on the advice of the UK Medicines Control Agency. 10 000 vials had already been shipped to 47 countries for use in lung scans. So far, Ireland is the only country to inform patients--because the Minister of Health, Brian Cowen, feels that people have a right to know. Countries such as the UK, Denmark, and Sweden have decided not to tell patients because the risk of transmitting nvCJD is thought to be negligible, and no test or prophylactic treatment is currently available.

On Dec 12, the Minister made the decision that the recipients should be told in a prudent and sensitive manner. Experts then began planning how to notify patients without causing panic. Instead, the media broke the story on the evening of Dec 14, causing worry and concern among all those who had had scans in the nine hospitals involved between July and mid-November.

Well worth noting: selected abstracts

 Failure to confirm a TSE in chickens [letter]
 Vet-Rec. 1997 Aug 23; 141(8): 203
 Cawthorne-RJ
This may have to due with controversial chickens submitted by Narang. Although birds have a well-characterized homologous prion protein, there has not been a serious study of their susceptibility to either mammalian or avian TSE. This is a sensitive area because many countries are diverting BSE and scrapie high-risk materials to chickens and pigs; mainstream researchers seek to avoid controversy. -- webmaster.
Post-exposure prophylaxis after accidental prion inoculation.
Lancet 1997 Nov 22;350(9090):1519-1520   No abstract
Aguzzi A, Collinge J
Detection of scrapie agent in the peripheral nervous system of a diseased sheep.
Neurobiol-Dis. 1996; 3(3): 191-5
Groschup-MH; Weiland-F; Straub-OC; Pfaff-E
In an attempt to determine whether scrapie infectivity can be found in the peripheral nervous system of a scrapie-diseased sheep, mice were inoculated intracerebrally or intraperitoneally with 10-fold dilutions of homogenates of Nervus (N.) axillaris, N. ulnaris, N. medianus, N. ischiadicus, N. tibialis, N. fibularis, and N.saphenus. Mice were observed for clinical signs of scrapie for 700 days and their brains were analyzed for accumulation of pathological prion protein by immunoblot. Substantial amounts of infectivity were found in all peripheral nerves tested except N.saphenus. Infectivity at titers of approximately 10(4.5) mouse infectious units (MIU)/g were detected in N. axillaris and N. ischiadicus, of approximately 10(3.0) MIU/g in N. ulnaris, N. medianus, N. tibialis, and N.fibularis, and of 10(6) MIU/g in the cerebellum. Since muscles are traversed by the nerve tracts tested, mutton of scrapie-diseased animals should not be regarded as being free of scrapie agent.
Differential allelic expression of PrP mRNA in carriers of the E200K mutation.
Neurology. 1997 Sep; 49(3): 851-6
Rosenmann-H; Halimi-M; Kahana-I; Biran-I; Gabizon-R
CJD linked to E200K presents with a wide range of age at disease onset. Since most patients are heterozygous for the mutation, we tested whether differential expression of mutant versus wild-type PrP may affect the age at disease onset in carriers of the mutation. Our results suggests that while in most healthy carriers the expression of wt PrP was higher than that of E200K PrP, most of the E200K CJD patients express equal levels of both PrP proteins. Similar results were obtained for both PrP protein and mRNA.

These results suggest that preferential expression of PrP from the wt allele may delay onset of the disease in carriers. This notion is consistent with the results obtained in transgenic mice carrying a human PrP gene, which suggest that endogenous PrP protects mice from contracting scrapie after inoculation with human CJD brain. Similar mechanisms may prevail in other inherited diseases with variable phenotypes.

Engineering peptides and proteins that undergo alpha-to-beta transitions.
Curr-Opin-Struct-Biol. 1997 Aug; 7(4): 501-8
Mihara-H; Takahashi-Y
In the 'protein-only' hypothesis, prion diseases are proposed to be the result of conformational changes of the normal form of the prion protein (PrPC) with a highly alpha-helical conformation to a pathogenic scrapie form (PrPSc) with a predominantly beta-sheet conformation. Recent studies examining the folding process of proteins, as well as the amyloidogenesis of peptides and proteins including prion proteins, Alzheimer's beta-peptides and other pathogenic protein mutants, have provided insight into the conformational changes essential to fibrillogenesis and correct folding.
Models of amyloid seeding in Alzheimer's disease and scrapie
Annu-Rev-Biochem. 1997; 66: 385-407
Harper-JD; Lansbury-PT Jr
Ordered protein aggregation in the brain is a hallmark of Alzheimer's disease and scrapie. The disease-specific amyloid fibrils comprise primarily a single protein, amyloid beta, in Alzheimer's disease, and the prion protein in scrapie. These proteins can be induced to form aggregates in vitro that are indistinguishable from brain-derived fibrils. Consequently, much effort has been invested in the development of in vitro model systems to study the details of the aggregation processes and the effects of endogenous molecules that have been implicated in disease. Selected studies of this type are reviewed herein. A simple mechanistic model has emerged for both processes that involves a nucleation-dependent polymerization. This mechanism dictates that aggregation is dependent on protein concentration and time. Furthermore, amyloid formation can be seeded by a preformed fibril. The physiological consequences of this mechanism are discussed.
The D178N (cis-129M) "FFI" mutation: diverse clinicopathologic phenotypes in Australian kindred.
McLean-CA; Storey-E; Gardner-RJ; Tannenberg-AE; Cervenakova-L; Brown-P
Neurology. 1997 Aug; 49(2): 552-8
Fatal familial insomnia (FFI) is an inherited prion disease characterized by progressive insomnia and dysautonomia with only modest cognitive impairment early in the disease, associated with atrophy and gliosis in the medial thalamus, but without spongiform change. FFI is associated with an aspartic acid to asparagine mutation at codon 178 of the PrP gene (D178N) in conjunction with methionine at the codon 129 polymorphic site on the mutant allele (cis-129M). We report a pedigree with this genotype in which marked clinicopathologic phenotypic heterogeneity occurred including typical CJD, FFI, and what was thought to be an autosomal dominant cerebellar ataxia (ADCA)-like-illness, suggesting that the genotype-phenotype correlation is not as tight for this mutation as is frequently supposed.
Familial prion disease with a novel 144-bp insertion in the prion protein gene in a Basque family.
Capellari-S; Vital-C; Parchi-P; Petersen-RB; Ferrer-X; Jarnier-D; Pegoraro-E; Gambetti-P; Julien-J
Neurology. 1997 Jul; 49(1): 133-41
Three members of a Basque family carrying a novel six R2 octapeptide repeat 144-bp insertion in the prion protein gene (PRNP) showed a slowly progressive dementia associated with cerebellar signs, myoclonic jerks, and seizures. Although postmortem examination revealed only focal and minimal spongiform degeneration in one subject with a 4-year course, significant astrogliosis and neuronal loss were associated with pronounced spongiform degeneration in the patient with a duration of symptoms of 10 years. Prion protein (PrP)-immunoreactive patches with a unique morphology were present in the molecular layer of the cerebellum in both subjects.

Western blot analysis demonstrated the presence of protease-resistant prion protein with the same size and ratio of the three differently glycosylated isoforms of that found in typical sporadic CJD129M/M. The amount of PrPres correlated with presence and severity of spongiform degeneration in the cerebral cortex. The findings suggest that a relatively low rate of PrPres deposition is the cause of the lack of spongiform degeneration in subjects carrying a 144-bp insertion in PRNP. The presence of PrP-immunoreactive patches with unique morphology in the molecular layer of the cerebellum is a hallmark of insertional mutations and is useful in the diagnosis of this subtype of human prion disease.

Interaction of 68-kDa TAR RNA-binding protein and other cellular proteins with prion protein-RNA stem-loop.
Scheffer-U; Okamoto-T; Forrest-JM; Rytik-PG; Muller-WE; Schroder-HC
J-Neurovirol. 1995 Dec; 1(5-6): 391-8
The RNA stem-loop structure of the trans-activating region TAR sequence of human immunodeficiency virus-1 mRNA is the binding site for a number of host cell proteins. A virtually identical set of proteins from HeLa nuclear extracts was found to bind to the predicted RNA hairpin element of prion protein (PrP) mRNA, as demonstrated in UV cross-linking/RNase protection and Northwestern assays. We show that the cellular TAR loop-binding protein, p68, is among those proteins which associate with PrP RNA. Competition experiments with various TAR RNA mutants revealed that binding of partially purified p68 to PrP RNA stem-loop occurs sequence-specifically. The 100-kDa 2-5A synthetase which is involved in the cellular antiviral defense was able to bind to PrP mRNA stem-loop in Northwestern blots with cytosolic proteins from HeLa cells treated with interferon. However, the PrP RNA failed to activate this enzyme in vitro, in contrast to TAR RNA.

Filaments formed by a leucine-rich repeat peptide: similarity to amyloid fibrils of prions and Alzheimer's.

Symmons-MF; Buchanan-SG; Clarke-DT; Jones-G; Gay-NJ
FEBS-Lett. 1997 Jul 28; 412(2): 397-403
We showed previously that a 23 amino acid peptide constituting a single leucine-rich repeat (LRRN) polymerises spontaneously in solution to form long filaments of a beta-sheet structure, a property similar to that of Alzheimer's beta-amyloid and prion peptides. Here we report that a variant of LRRN in which a highly conserved asparagine residue is replaced by aspartic acid does not form either filaments or beta structure. By contrast, a variant which replaces this asparagine residue with glutamine forms filaments ultrastructurally indistinguishable from those of LRRN.

Electron micrographs of LRRN filaments show that many consist of two interleaved strands which appear to have a ribbon-like morphology. X-ray diffraction patterns from oriented LRRN fibres reveal that they are composed of long beta-sheet arrays, with the interstrand hydrogen bonding parallel to the filament axis. This 'cross-beta' structure is similar to that adopted by beta-amyloid and prion derived fibres. Taken together, these results indicate that the LRR filaments are stabilised by inter- or intra-strand hydrogen bonded interactions comparable to the asparagine ladders of beta-helix proteins or the 'glutamine zippers' of poly-glutamine peptides.

We propose that similar stabilising interactions may underlie a number of characterised predispositions to neuro-degenerative diseases that are caused by mutations to amide residues. Our finding that amyloid-like filaments can form from a peptide motif not at present correlated with degenerative disease suggests that a propensity for beta-filament formation is a common feature of protein sub-domains.

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