October 1999 Prion Science News
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Doppel disease
CWD: not attributable to 129L allele in elk
CWD: oral transmission to fawns
Detection of BSE-specific rogue prion by heat and guanidine thiocyanate
Raman pH dependence of copper binding to prion octapeptide
Monoclonal antibody philosophy
More from Tubingen meeting of Sept 1999
Prion glycoform varies by brain region
Large new kindred of A117V
Familial dementia: loop insertion polymerization of neuroserpin
Hereditary cardiac amyloidosis

Doppel disease

J Mol Bio 1 Oct 99 292 797-817
RC Moore, IY Lee, GL Silverman, PM Harrison, RStrome, C Heinrich, A Karunaratne, SH Pasternak, MA Chishti, Y. Liang, P Mastrangelo, K Wang,  AFA Smit, S Katamine, GACarlson, FE Cohen, SBPrusiner, DWMelton, P Tremblay, LE Hood, D Westaway
The discovery of a late-onset ataxia with Purkinje cell degeneration in mice with upregulated doppel gene is a paper-within-a-paper. The discovery came about in trying to reconcile conflicting reports concerning the effect, or lack or effect, of prion gene knockouts in various strains of mice.

The marked evolutionary conservation of prion protein sequence suggested an essential function, so lack of an effect in a knockout beginning either at embryogenesis or in adulthood suggested additional, compensatory proteins. By April 1995, a tandem paralogue, doppel, was identified. It was expressed as mRNA in normal mice during embryogenesis but in adult mice was expressed at high levels in testes and heart but not lung and at low but definite levels in brain, ie, its tissue-specific regulation differed from prion protein. (The EST collection at GenBank greatly expands the tissue set with doppel expression.)

Doppel also competed for the prion exon 2 splice site with prion pre mRNA (ie, the ancient tandem duplication was downstream of prion promoter). This, and its structural homology, made it a prime candidate for a compensatory protein. In fact, disparities in mouse strains correlated perfectly with doppel mRNA upregulation of the exon 2 type in strains developing ataxia (which amounts to over-compensation).

Note that over-production of doppel mRNA in brain homogenate is not at all the same thing as over-production of doppel protein in Purkinje cells. Even when the exon 3 splice acceptor is missing, the poly A signal and site are still intact, meaning that a curious prion mRNA consisting of exon 1 and 3' UTR of exon 3 could still be produced. Anti-prion transcripts, as studied by Oesch et al. are still produced in prion gene deletions of Zurich I type. Now doppel coding mRNA aligns poorly with prion in vitro and in silico, so anti-sense transcripts of doppel are doubtful. This leaves open the question as to what part of the genome is giving rise to anti-prion transcripts and what, if anything, this has to do with regulation of either gene or the ataxia.

Paralogue compensation effects are common in hemoglobinopathies.

Knockouts in mammals are scarcely precision coding deletions of the type familial from yeast and E. coli and often exhibit major extraneous effects. Here, the ataxic deletions extended 1 kb upstream from the ORF, thus taking away the prion gene's own splice acceptor, leaving no choice but for prion pre mRNA to mature into doppel mRNA. While an artefact in the Japanese strains, this deletion would have been a natural one to construct given the chimeric transcripts.

Since tissue-specific expression in adult brain normally favors copious expression prion protein in conjunction with low alternative splicing to doppel, this meant high levels of doppel mRNA were now inappropriately produced in brain.

Despite an antibody to synthetic doppel peptide, CDIDFGAEGNRYYAANYWQFPD, no study was made of doppel protein abundance. Even if this correlated perfectly with mRNA levels, no mechanism is at hand for doppel protein excess to directly cause the ataxia -- doppel has completely lost the amyloidogenic region. Purkinje cells may well localize the problem but how is the mechanism for cell death to be established? It was not actually shown here that any doppel mRNA was produced in affected Purkinje cells nor what background level of doppel mRNA Purkinje cells have (brain homogenate was low but perhaps some cell types are high).

OMIM lists 386 entries for ataxias -- what if anything does the doppel-associated ataxia have to do with ataxia found sometimes in prion disease? Conceivably, some of these ataxias could result from alterations in the exon 3 splice site. [Note that the -21 A/G allele associated with A117V is a candidate for altering levels of chimeric transcripts.]

Getting back to prion gene knockouts that don't delete the prion gene splice acceptor, doppel production from its own promoter could still be compensatory. Indeed, all the known mutations in the prion CJD apparently affect amyloidogenicity, not normal function. It is clear that all the various combinations of single and double deletions (and point mutations) will need to be constructed and characterized, in the presence and absence of infection, in cis and in trans.

Doppel mutations may cause human diseases of its own -- these need not be neurological (eg, infertility due to non-expression in testes). Zitter rats and a human neurological disorder with normal prion gene but chr 20 12pter location are early candidates. Without amyloid, the mechanism for autosomal dominance is lost -- it is much harder to detect rarer autosomal recessive disease. However, some Purkinje cell loss is reported in heterozygotes, so overproduction by whatever mechanism may eventually manifest itself as ataxia.

The primate and artiodactyl lineages may or may not have retained chimeric splicing. Humans have certainly retained an active doppel gene and promoter; however, prion exon 2 is so far cryptic. Nonetheless, discovery of the doppel gene will require re-examination of sporadic CJD, scrapie and CWD alleles, and certainly the interplay of doppel and prion in BSE and nvCJD susceptibility.

The experimental discovery and characterization of dopple gene, its coupled regulation, and protein structure truly open a new era. This is a very substantial paper with thorough experimental support for its conclusions.

But one wonders what is to be done with data from the old era. For example, knockout mice are said not be susceptible to TSE -- how did the particular knockout affect doppel gene expression? Mice with knock-in human prions are said to test BSE susceptibility of humans but did the remaining mouse doppel protein interface normally with human prion protein?

Perhaps there is a double species barrier; prion-prion interference is already common in TSE across species and within heterozygotes. If the infecting prion cannot interact properly with host doppel protein as well as host prion protein, this could mean nvCJD studies have dramatically understated the risk as investigators did not knock in a human doppel as well. Nothing is none at this time about bovine or ovine doppel proteins or their interactions with host prions.

PrP's Double Causes Trouble

29 Oct 99 Science
C Weissmann and A Aguzzi
Comment (webmaster): This is a mainstream commentary piece that accurately recapitulates the JMolBio findings and provides a re-analysis of ataxia in an older Zurich 1 prion CDS truncation carried on a transgene construct not expressed in Purkinje cells and also the unpublished outcome of what happens when this is fixed. The lesson here is that transgenic mice need to be characterized very carefully.

Highlights:
"Why should overexpression of Dpl cause ataxia and concurrent overexpression of PrP restore normal function? Shmerling et al. (12) found that introduction into Zürich I Prnp0/0 mice of a truncated Prnp transgene lacking the amino terminus and, hence, devoid of the octarepeats and the conserved 106-126 amino acid region (PrPD32-135) resulted in ataxia and degeneration of the cerebellar granule cell layer within weeks after birth.

Moreover, introduction of a single intact PrP allele prevented the disease. They proposed that PrP interacts with a ligand to elicit an essential signal and that a PrP-like molecule with lower binding affinity could fulfill the same function in the absence of PrP. According to this hypothesis, in PrP knockout mice the truncated PrP could interact with the ligand, displacing the PrP-like molecule, but without eliciting the survival signal. If PrP has the higher affinity for the ligand, it could displace its truncated counterpart and restore function.

Because Dpl resembles truncated PrP, it might cause disease by the same mechanism, namely by competing with PrP for the PrP ligand. However, because the promoter used to express the truncated PrP is active in granule cells but not in Purkinje cells, whereas the wild-type PrP promoter is active in Purkinje cells, the cells targeted in mice expressing truncated PrP may be different from those in Nagasaki mice expressing Dpl. Indeed, targeting the truncated PrP to Purkinje cells causes them to die, leading to ataxia (13).

The discovery of PrP's double solves some questions but raises others. Evidence for the expression, proposed structure, and cellular location of the predicted Dpl protein is still lacking. Will transgenes encoding Dpl indeed cause degeneration of cells in which they are overexpressed? Does the time of appearance and intensity of the pathological phenotype depend on the ratio of Dpl to PrP? What is the physiological function of Dpl, and is it important in prion diseases? Are there genetic defects in humans elicited by overexpression of Dpl in the brain? And, now that we know that PrP is not required to maintain Purkinje cell function, what then is its purpose? But perhaps one of the most important lessons from these findings is that disrupting a gene may entail far more than the phenotype that meets the eye."

13.E. Flechsig, R. Leimeroth, C. Weissmann, unpublished data.
12.D. Shmerling et al., Cell 93, 203 (1998) [Medline].

Expression of amino-terminally truncated PrP in the mouse leading to ataxia and specific cerebellar esions.

Cell 1998 Apr 17;93(2):203-14
Shmerling D, Hegyi I, ... Hangartner C, Aguzzi A, Weissmann C
The physiological role of prion protein (PrP) remains unknown. Mice devoid of PrP develop normally but are resistant to scrapie; introduction of a PrP transgene restores susceptibility to the disease. To identify the regions of PrP necessary for this activity, we prepared PrP knockout mice expressing PrPs with amino-proximal deletions. Surprisingly, PrP lacking residues 32-121 or 32-134, but not with shorter deletions, caused severe ataxia and neuronal death limited to the granular layer of the cerebellum as early as 1-3 months after birth. The defect was completely abolished by introducing one copy of a wild-type PrP gene. We speculate that these truncated PrPs may be nonfunctional and compete with some other molecule with a PrP-like function for a common ligand. [The authors already knew about doppel at the time this article was written. -- webmaster]

Oral transmission and early lymphoid tropism of chronic wasting disease PrPres in mule deer fawns (Odocoileus hemionus)

J Gen Virology Oct 1999 pg 2757-2764 free pdf  [submitted on 23 Mar 99]
 C. J. Sigurdson, E. S. Williams, M. W. Miller, T. R. Spraker, K. I. O'Rourke and E. A. Hoover
This paper establishes very effective oral transmission of CWD to mule deer. Protease K -resistant prions were detected as early as 42 days post-inoculum in the tonsil, Peyer's patch, retropharyngeal lymph node, and ileocaecallympth node in all fawns examined subsequently, ie, in lymphoid tissues draining the alimentary tract. No prions were detected in neural tissue; conversely, intra-cerebrally control-inoculated fawns had no prions in their lymph system. The experiment terminated without the intent of seeing whether affected fawns went on to develop clinical CWD.

This is very bad news because of the rapid and efficient transmission of CWD by oral route (though animals would scarcely be exposed to pure CWD brain in the wild). The study does not prove that oral transmission is the principal route of horizontal transmission but certainly suggests this. [Protease K resistance is also just a proxy for infectivity.]

The authors report a major improvement in detection methodology: sequential tissue treatment by formic acid, protease K, hydrated autoclaving, followed by immunohistochemical staining with monoclonal antibody F89/160.1.5 (which they have made commercially available). Figure 1c shows the dramatic improvement in stainability of tonsillar llymphoid follicles.

Mice are said to be relatively resistant to CWD transmission [M. Bruce, pers. comm]. Here the fawns were repeatedly exposed over 5 days. The inoculum was prepared in normal saline from the brains of 26 deer with confirmed CWD.

One cause for concern is the statement that two of the "negative" control fawns (taken from a wild population not known to have CWD) showed a greyish pink stain in as many as 5 follicles. These could very well represent early incipient CWD in these animals because 46 other control deer lacked them. A more extensive follow-up of tissues from these two "negative" fawns is needed.

The results here are very much in line with what is seen in humans, monkeys, cows, sheep,goats, and rodents exposed to oral doses of various TSEs, although in deer the prions are disseminated throughout lymphoid tissues. These lymphoid tissues are adjacent to mucosal surfaces that made shed agent into saliva and faeces, infecting other animals(inlcuding livestock) inadvertantly grazing upon them.

What is remarkable here is the extraordinary early detection using the improved method. This may be partly a consequence of the 5 days of sequential exposure but the improved assay method is clearly a major improvement in detecting early CWD.

PrP genotypes of captive and free-ranging Rocky Mountain elk (Cervus elaphus nelsoni) with chronic wasting disease

J Gen Virology Oct 1999 pg 2765-2769 free pdf
 K. I. O'Rourke, T. E. Besser, M. W. Miller, T. F. Cline, T. R. Spraker, A. L. Jenny, M. A. Wild, G. L. Zebarth and E. S. Williams
CWD probably arose in confined cervids as a horizontal transfer of scrapie from sheep co-housed and pastured previously at the Dept of Wildlife's Foothills Research Station in Ft. Collins, Colorado. This study looked at whether a coding polymorphism in elk (M129L) could have been responsible instead. Ironically, the polymorphism turned out to be protective.

In 387 carefully sequenced elk, only this one coding polymorphism was observed. A silent change was also seen in codon 104, namely AAG to AAA. This region is not within hairpin C. (Flanking primer pairs were not used after learning a mule deer allele was not amplified; that allele is not described. ) Genotype frequencies were 0.75, 0.24, and 0.01, for met/met, met/leu, and leu/leu, ie, in approximate Hardy-Weinberg equilibrium with 12% leucine. This is not a possible frequency for a mutation that could cause disease onset in a 2 year old elk; however, it might well affect susceptibility, as seen in human CJD.

Met/met was signficantly over-represented in CWD-affected game farm animals at the South Dakota facility. CWD was seen in some heterozygotes (without a striking delay in onset) but not in leucine homozygotes.

The results are difficult to interpret. The genotype of the original infecting animal(s) is not known. Sheep of course are met/met, as are mule deer. Assuming it was met/met, then the like-like principle favors transmission to similar animals and a barrier might occur to unlike animals. This is like the situation in BSE, where met/met cows are initially infecting met/met humans instead of val/val. A leu/leu elk with CWD may eventually be found; that animal might conversely transmit better to other leu/leu animals.

The study did rule out "familial CWD" involving 129L viewed as a rare mutation. However, some other rare mutation, long since lost, could have initiated the epidemic. Non-coding mutations, such as upregulation alleles linked to met, could also be a factor. Other genes, such as cervid doppel, were not examined in this study.

The South Dakota game farm is severely impacted with 42 confirmed cases dating back to 1995, strongly suggesting horizontal efficient transmission. Leakage from this facility is of grave concern to large herds of wild elk on surrounding public and tribal lands. While no CWD was reported in pen mates or via fenceline contact in this study, a previous study has documented transfer from captive elk to wild deer across a fenceline.

When no CWD is detected, one always has to wonder if the incubation period was too short or whether the methods were not sensitive enough. This study used good methods for their day, but not the ultra-sensitive methods that would be used now. For that reason, negative results must be revisited and for now regarded with caution. The vastly improved detection method of the companion paper was not used: sequential tissue treatment by formic acid, protease K, hydrated autoclaving, followed by immunohistochemical staining with monoclonal antibody F89/160.1.5.

The Montana facility is also reported negative. Since the paper was submitted, a second elk shipped to Oklahoma was confirmed positive. The Montana Wildlife Federation has also strongly questioned testing procedures at the farm; an elk with a missing head was found.

The politics of CWD is explosive: it is bad enough to have the disease endemic in Rocky Mtn National Park -- to have it escape from already-controversial game farms into huge elk populations at Yellowstone NP and the Black Hill, not to mention cattle and sheep on public and private lands, is almost unthinkable.

Two of the authors of this paper draw their salaries primarily through the sale of Colorado elk hunting tags and a third is an employee of the game farm industry. Indeed, it is impossible to obtain necessary CWD samples without the cooperation of their host institutions. This paper however appears scientifically sound and does not exhibit much by way of overt bias.

Detection of BSE-specific rogue prion by treatment with heat and guanidine thiocyanate

Journal of Virology, November 1999, p. 9386-9392, Vol. 73, No. 11  [blocked]
 Rudolf K. Meyer,Bruno Oesch,  Rosmarie Fatzer,  Andreas Zurbriggen,  and Marc Vandevelde
The conversion of a ubiquitous cellular protein (PrPC), an isoform of the prion protein (PrP), to the pathology-associated isoform PrPSc is one of the hallmarks of transmissible spongiform encephalopathies such as bovine spongiform encephalopathy (BSE). Accumulation of PrPSc has been used to diagnose BSE. Here we describe a quantitative enzyme-linked immunosorbent assay (ELISA) that involves antibodies against epitopes within the protease-resistant core of the PrP molecule to measure the amount of PrP in brain tissues from animals with BSE and normal controls. In native tissue preparations, little difference was found between the two groups.

However, following treatment of the tissue with heat and guanidine thiocyanate (Gh treatment), the ELISA discriminated BSE-specific PrPSc from PrPC in bovine brain homogenates. PrPSc was identified by Western blot, centrifugation, and protease digestion experiments. It was thought that folding or complexing of PrPSc is most probably reversed by the Gh treatment, making hidd

en antigenic sites accessible.

The digestion experiments also showed that protease-resistant PrP in BSE is more difficult to detect than that in hamster scrapie. While the concentration of PrPC in cattle is similar to that in hamsters, PrPSc sparse in comparison. The detection of PrPSc by a simple physicochemical treatment without the need for protease digestion, as described in this study, could be applied to develop a diagnostic assay to screen large numbers of samples.

Scrapie Pathogenesis in Subclinically Infected B-Cell-Deficient Mice20

Journal of Virology, November 1999, p. 9584-9588, Vol. 73, No. 11 [blocked]
Rico Frigg,Michael A. Klein, Ivan Hegyi, Rolf M. Zinkernagel, and Adriano Aguzzi
Prion infections can present without clinical manifestations. B-cell deficiency may be a model for subclinical transmissible spongiform encephalopathy, since it protects mice from disease upon intraperitoneal administration of scrapie prions; however, a proportion of B-cell-deficient mice accumulate protease-resistant prion protein in their brains. Here, we have characterized this subclinical disease. In addition, we have studied the possibility that a neurotoxic factor secreted by B cells may contribute to pathogenesis.

Raman spectroscopy of pH dependence of copper binding to prion octapeptide

Biochemistry 1999 Aug 31;38(35):11560-9
Miura T, Hori-i A, Mototani H, Takeuchi H
This paper, which looked from the abstract just to be the nth variation on how copper binds to the prion repeat structure, is actually quite interesting in the full text. They did a nice job of reconciling their data with several previous studies, some of which were conflicting.

Note than prion doppel lacks anything resembling a copper-binding tandem repeat or any facility for binding multiple coppers. There is no experimental data on cofactor binding. This raises the questions of what doppel is doing and whether the copper site has been lost in the doppel pedigree or acquired in that of the prion. In Brown's model for a superoxide dismutase, doppel would be left sitting on the sidelines.

Hornshaw et all got copper repeat structure off to a good start back in a 15 Feb 95 BBRC article, but an appalling lack of progress followed. A model by Stockel J et al. [Biochemistry. 1998 May 19;37(20):7185-93] made good structural sense but it was soon contradicted (without explanation) by another paper from the same lab, Viles JH et al. [PNAS1999 Mar 2;96(5):2042-7 (which is oddly misplaced by Medline)].

The 3 issues addressed here are how many copper atoms are bound per tetrarepeat, what are their amino acid ligands, and how do these change with mildly lowered pH. (Zinc has dropped off the radar screen without explanation and no one wants to synthesize a full repeat domain.)

Miura et al. conclude from Raman stretching frequencies that ring nitrogen of histidine closest to the beta carbon (pi nitrogen), plus the second and third deprotonated glycine amide nitrogens of PHGGGWGQ do the copper binding at neutral and basic pH. At weakly acidic pH 6.0, these amide nitogens are protonated and the histidine binding changes to the distal ring nitrogen (tau), causing the stoichiometry to drop from 4 to 2 coppers bound and onset of inter-molecular copper cross-bridging and aggregation. Thus the discrepancy between previous equilibrium dialysis experiments is simply attributable to pH.

They suggest the aggregation could result in vivo during endosomal comparmentalization; though repeat region is often cleaved off of amyloid, the copper has at least brought monomers together in close proximity. This does not explain why the disease is so rare nor why extra repeat insertions are disease-causing.

On the other hand, they note that the 20 mM peptide concentration is not found in vivo, suggesting that the conformational change with pH may be intra-molecular. This would have the effect of giving the repeat region a 'grab and drop' cycle, where, say, the long arm reaches out to protect the synapse by soaking up 4 copper atoms, then drops them inside as the pH changes in the cycling noted by Harris et al. While others have considered this (eg, http://www.mad-cow.org/prion_proline.html ), the paper here supplies a rationale and details of the pH 6.4 switch at the atomic level. This is surely not the primary mode of copper transport in mammalian cells and would be more of a detoxification step than cell nutrition-related.

Suppose that scenario is valid. Why would a prion protein knockout with accompanying upregulation of doppel be associated with ataxia but no accompanying upregulation not be associated with ataxia? If doppel cannot bind copper, it seems like there should be copper damage to the synapse in either situation. If doppel is somehow a primitive copper shuttle, the ataxia is backwards: the non-upregulated mouse line should be affected.

A nice discussion is given of the analogous N-terminal copper binding sites in serum albumins and the gly-gly-his model peptide literature. In short, mammalian prion copper binding site chemistry and geometry is nothing new at the local level.

Raman spectroscopic study on the Copper(II) binding mode of prion octapeptide and its pH dependence

. The cellular form of prion protein is a precursor of the infectious isoform, which causes fatal neurodegenerative diseases through intermolecular association. One of the characteristics of the prion protein is a high affinity for Cu(II) ions. The site of Cu(II) binding is considered to be the N-terminal region, where the octapeptide sequence PHGGGWGQ repeats 4 times in tandem. We have examined the Cu(II) binding mode of the octapeptide motif and its pH dependence by Raman and absorption spectroscopy. At neutral and basic pH, the single octapeptide PHGGGWGQ forms a 1:1 complex with Cu(II) by coordinating via the imidazole N(pi) atom of histidine together with two deprotonated main-chain amide nitrogens in the triglycine segment. A similar 1:1 complex is formed by each octapeptide unit in (PHGGGWGQ)(2) and (PHGGGWGQ)(4).

Under weakly acidic conditions (pH approximately 6), however, the Cu(II)-amide(-) linkages are broken and the metal binding site of histidine switches from N(pi) to N(tau) to share a Cu(II) ion between two histidine residues of different peptide chains. The drastic change of the Cu(II) binding mode on going from neutral to weakly acidic conditions suggests that the micro-environmental pH in the brain cell regulates the Cu(II) affinity of the prion protein, which is supposed to undergo pH changes in the pathway from the cell surface to endosomes. The intermolecular His(N(tau))-Cu(II)-His(N(tau)) bridge may be related to the aggregation of prion protein in the pathogenic form.

Evidence of Presynaptic Location and Function of the Prion Protein

Journal of Neuroscience, October 15, 1999, 19(20):8866-8875 [blocked]
Jochen Herms, ..., and Hans Kretzschmar 
The prion protein (PrPC) is a copper-binding protein of unknown function that plays an important role in the etiology of transmissible spongiform encephalopathies. Using morphological techniques and synaptosomal fractionation methods, we show that PrPC is predominantly localized to synaptic membranes. Atomic absorption spectroscopy was used to identify PrPC-related changes in the synaptosomal copper concentration in transgenic mouse lines. The synaptic transmission in the presence of H2O2, which is known to be decomposed to highly reactive hydroxyl radicals in the presence of iron or copper and to alter synaptic activity, was studied in these animals.

The response of synaptic activity to H2O2 was found to correlate with the amount of PrPC expression in the presynaptic neuron in cerebellar slice preparations from wild-type, Prnp0/0, and PrP gene-reconstituted transgenic mice. Thus, our data gives strong evidence for the predominantly synaptic location of PrPC, its involvement in the regulation of the presynaptic copper concentration, and synaptic activity in defined conditions.

Monoclonal antibodies specific for the native, disease-associated isoform of the prion protein.

Methods Enzymol 1999;309:106-22  [offline]
Korth C, Streit P, Oesch B   [UCSF]
 
Reviewing the circumstances that have led to the first monoclonal antibody against the disease-associated form of PrP, we consider the availability of PrP knockout mice and recombinant PrP, as well as a reliable conformational screening protocol as being important prerequisites for a successful immunization approach.

When considering presenting an antigen to a mouse with the goal of obtaining specific monoclonal antibodies against a misfolded or aggregated form of a host protein, it is desirable to increase the definition of a subtle conformational difference. This can be achieved by immunizing an antigen knockout mouse that has not developed self-tolerance against the respective antigen.

Furthermore, if conformational isoforms and/or oligomeric forms of a protein sequence are understood to exist in an equilibrium, high and pure amounts of recombinant protein may increase the likelihood that a particular population of protein conformation passes an antigenic threshold necessary to start an immunogenic response.

Pulling out the monoclonal antibodies by correct screening is essential. Screening against the pure misfolded or aggregated protein is often complicated by its poor solubility and hence the ability to immobilize. In the present case, immobilization of disease-associated PrP on nitrocellulose had been established as a conformation-sensitive screening method, allowing to "freeze" PrP in its distinguishable, disease-associated conformation.

We are cautious to generalize conclusions of how to assess the generation of monoclonal antibodies against these particular protein isoforms to other diseases of protein misfolding and/or aggregation, but ultimately the present approach may inspire respective experiments.

Comment (webmaster): The abstract -- a turgid philosophical tract --does not make clear whether they have new monoclonals or not. The proof is in the pudding.

More from Tubingen meeting

Correspondents in Europe Sept 28, 1999
What attendees wrote the webmaster about the major TSE meeting this fall in Tubingen, Germany and some bracketed commentary:

"Not much spectacular news. Some talks were only reviews of 3 year old published data. Poster sessions were better (over 160) but time for these was very short (3 x 20minutes). [Which works out to 23 seconds per poster.]

A proceedings containing more or less all speakers talks will be available at some unknown date. [No doubt off-line, expensives, minimally edited, and obsolete by the time it appears.]

Opening lecture of C. Weissmann: their original knockout mice (designated ZH1) had no Purkinje cell loss, whereas the Japanese kockout had considerable Purkinje cell loss. Zurich then made new knockout mice, ZHII, also exhibiting Purkinje cell loss. The difference turned out to be that ZHI mice had Neo insertion in full PrP ORF containing exon, while Japanese strain and lost the 5' end of this exon. The ZHII mice are also missing the 5' of this exon. Crossed mice (ZHII/ZHI) had significant less Purkinje cell loss than ZHII, but more than ZHI. Their hypothesis was: signals in the 5' half of exon III normally downregulate expression of dpl (Prn-d). Both ZHII and RCN0 have overexpression of dnpl leading to ataxia.

- Most of the people seem convinced of the nucleated polymerisation mechanism for prion fibril formation. [Wake me when its over]

- {A. Aguzzi} IL-6 deficiency in mice renders these mice more susceptible to prions. [interleukin cytokine, 16000 medline hits]

- {D. Dormont} Congo red makes mice PrP-Sc more PK resistant but not hamster. [Intercalation not going to be a cure-all, might make it worse.]

- {L. van Keulen} Sequential timepoint study; ENS acts as port of entry. [Typo for CNS? Lot of similar studies]

- {A. Buschmann} Bovine transgenic mice are susceptible to BSE. Chimeric mo-bo-mo Tg are resistant to BSE (junctions 1-94 mo, 95-188 bo, 189- mo). 2nd passage bovine transgenic mice decreases incubation time. bovine transgenic mice in chimeric background have incubation period of 250-350 days. [lot of similar studies]

- {MJ Schmerr} Lambs in scrapie environment positive in blood at 4 weeks of age (even one at 2 weeks!). Test works on elk, mule deer, sheep, hamster. [This is progress to have such an early diagnosis and suggests that sheep flocks can 'test out' of the disease, depending on how long it persists in pastures and paddocks.]

- {J. Wadsworth} EDTA and metal ions make switching between the currently defined glycoprofiles possible! Even the shift after PK digestion can be altered (19 or 20-21 kDa). Types 1 and 2 could be altered, types 3 and 4 remained unchanged. [Now this is both interesting and disturbing. Those bands were supposed to be fully denatured in SDS. Where would there be metal binding if there is removal of the repeat? Could not be inter-molecular copper as recently proposed. Must be intra-molecular domain that can still retain conformation in SDS in presence of metal, migrating slower.]

- {Wuthrich} Proposed that the N-terminal part of PrP-27-30 is the only buried conformational change. Alpha helices etc remain unchanged. [Right. The rest of the protein is just an appendage to the cross-beta just like it is in all the other amyloidoses. People screwed up interpretation of circular dichroism spectra, cross-beta comes in a confusing wevelength.]

- {B Caughey} More physiological conditions using NaCl/KCl conserve strain specificity. [Improvement on recent publication on this same topic? What has become of conversion of human by CWD deer and elk??? See:

Assays of protease-resistant prion protein and its formation.

Methods Enzymol 1999;309:122-33 [no abstract]
Caughey B, Horiuchi M, Demaimay R, Raymond GJ]
- {M Scott} bovine transgenic mice mice. heterozygous 8-fold overexpression. BSE transmits 1st pass 234+/-8 days (similar pathol), serial pass 217+/-6. vCJD transmits to these transgenics 247+/-4 days and Suffolk scrapie transmits to these Tg with large variation 203-245 days. Lesion profiles BSE, vJCD, Sc different. Histoblot vCJD, BSE identical, Sc different. vCJD+BSE plaque positive, Sc plaque negative." [No evidence here for scrapie as cause of BSE epidemic. Yet there are always more strains of scrapie out there to test.]

J Gen Virology has 2 further upcoming articles in Nov 99:

Pathogenesis of the oral route of infection of mice with scrapie and bovine spongiform encephalopathy agents
T. Maignien, C. I. LasmÈzas, V. Beringue, D. Dormont and J.-P. Deslys 3035 - 3042

Transmission of the 263K scrapie strain by the dental route
L. Ingrosso, F. Pisani and M. Pocchiari 3043 - 3047

Prion glycoform varies by brain region

J Neuropathol Exp Neurol 1999 Sep;58(9):1000-9 
DeArmond SJ, Qiu Y, Sanchez H, Spilman PR, Ninchak-Casey A, Alonso D, Daggett V
This paper sounds like a welcome addition to a long overdue explanation for phenotype heterogenity and strain types in TSEs. The problem has always been the considerable variability (60 intermediates) of the glycans, the difficulty of their covalent characterization even with the most modern methods, and the small amounts available (even less if particular brain regions are differentiated).

Charge differences here have to due with the number of tetra-antennary sialic acids; multiple species with the same charge (eg, three with charge -3) will sometimes have the same molecular weight, so are not resolvable in 2D gels and barely in GC-MS.

If the differances by brain region really scale at the level of neuronal cell type (which is a quite reasonable), how are homogeneous glycosylation regions to be dissected out in sufficient quantity for assay? Do glycosylation differences arise qualitatively (some enzyme at some stage of glycan building is turned on or off ) or only quantitatively (some enzyme at some stage of glycan building is turned on to a continuously variable degree ) ?

Studies of this type thus run into a brick wall.

Strain-typing has always been bothersome in that researchers are looking at an average over a brain sample large enough to be quite heterogeneous. In other words, the monomers of an individual amyloid fibril could be quite homogeneous (say one site always glycosylated with a single glycan species, the other site always vacant) but by the time this is homogenized in with fibrils from a thousand other cell types, it is a messy average over a lot of secondary biosynthetic (and catabolic) enzymes in a lot of cell types. This potentially brings in a large number of alleles of those secondary enzymes to account for individual differences, which are noted even in siblings in familial CJD. In this scenario, it is just luck if enough of a signature survives averaging to determine the route of infection (eg, dietary vs endogenous) or source of infection (eg, cow vs sheep).

Their hypothesis is reasonable: accumulation and the vacuolation pattern phenotypes in the brain are "governed" by neuron-specific differences in normal prion glycoforms, though it will be hard to go beyond strong correlation.

Until something better comes alone, the physical explanation would simply be the like-like principle in fibril crystal growth. This predicts that the recruiting prion looks for normal prions glycosylated like itself (just as a seed grain of table salt put in a saturated mix of sea salts looks for the sodium chloride). The prions like itself are distributed variably by brain region or age or allele genetics or whatever and so that is where the pathology occurs. However N- and C-terminal truncation may be just as important as the carbohydrate side chains.

PrPc glycoform heterogeneity as a function of brain region: implications for selective targetingof neurons by prion strains.

J Neuropathol Exp Neurol 1999 Sep;58(9):1000-9 
DeArmond SJ, Qiu Y, Sanchez H, Spilman PR, Ninchak-Casey A, Alonso D, Daggett V
We recently found that deletion of the Asn-linked carbohydrate (CHO) at residue 197 of Syrian hamster (SHa) PrP(C) while retaining the CHO at Asn 181 has a profound effect on which population of neurons are targeted for conversion of SHaPrP(C) to SHaPrP(Sc) in transgenic (Tg) mice inoculated with scrapie prions.

We hypothesized that selective targeting of neuronal populations is determined by cell-specific differences in the affinity of an infecting prion) for normal prion and that the affinity might be modulated by nerve cell-specific differences in the latter's glycosylation.

Here we tested this hypothesis by assessing whether or not each brain region in Syrian hamsters synthesizes different PrP(C) glycoforms, as inferred from 2D-gel electrophoresis. Reproducible differences in the number and isoelectric point of PrP(C) charge isomers were found as a function of brain region. The results of this study support the hypothesis that the PrP(Sc) accumulation and the vacuolation pattern phenotypes in the brain are governed by neuron-specific differences in PrP(C) glycoforms.

Inherited prion disease A117V in the prion protein gene

Brain, Vol. 122, No. 10, 1823-1837, October 1999 
G. R. Mallucci,T. A. Campbell, A. Dickinson, ..., P. Ince, J. W. Ironside, L. R. Bridges, A. Dean, I. Weeks and J. Collinge 
A large English family with autosomal dominant segregation of presenile dementia, ataxia and other neuropsychiatric features is described. Diagnoses of demyelinating disease, Alzheimer's disease, Creutzfeldt-Jakob disease (CJD) and Gerstmann-Str”ussler-Scheinker syndrome have been attributed to particular individuals at different times.

An Irish family, likely to be part of the same kindred, is also described, in which diagnoses of multiple sclerosis, dementia, corticobasal degeneration and new variant CJD have been considered in affected individuals. Molecular genetic studies have enabled the classification of this disease at the molecular level as one of the group of inherited prion diseases, with the substitution of valine for alanine at codon 117 of the prion protein gene (PRNP).

Only three other kindreds have been described world-wide with this mutation and only limited phenotypic information has been reported. Here we describe the phenotypic spectrum of inherited prion disease (PrPA117V). The diversity of phenotypic expression seen in this kindred emphasizes the logic of molecular classification of the inherited prion diseases rather than classification by specific clinicopathological syndrome. Indeed, inherited prion disease should be excluded by PRNP analysis in any individual presenting with atypical presenile dementia or neuropsychiatric features and ataxia, including suspected cases of new variant CJD.

Comment (webmaster): Previously described kindreds had a tightly linked allele at position -21 relative to the ATG start codon. Let's hope they looked at this in the kindred below and also determined whether or not the Irish kindred was an extension with microsatellite analysis. Strain type was surely determined; is it consistent across theis and other kindreds?

One sees 8 diagnoses scattered all over the clinical landscape that the family received over the years. This is not attributable so much to incompetency in neurology but rather to heterogeneity in presentation so characteristic of TSEs. This surely supports the authors' recommendation for using molecular classification. But where does that stop -- surely not with just the prion coding region..

Familial dementia caused by polymerization of mutant neuroserpin

23 September 1999 Nature 401, 376 - 379 (1999)
Richard L. Davis, Antony E. Shrimpton, ... & David A. Lomas
Comment (webmaster): This article, while a high quality one that resolved an autosomal dominant late onset dementia, was also the subject of a shabby press release yesterday from the journal. The new disease is not even an amyloidosis, so what relevence does it have to causation of phenotype and therapy in AD and CJD?

The key claim -- that the protein fibril per se causes the damage -- had strictly circumstantial supporting evidence and certainly was not resolved. Without really doubting the conclusion (here or in CJD), when nothing is offered by way of a polymer pathogenic mechanism, other mechanisms of toxicity really have to be ruled out, especially in a protein that by definition inhibits other neuroproteins.

Still, this is a very interesting class of disease, related to loop-sheet polymerization, which is an intrinsic flaw to the design of a large superfamily of proteins (serpins) when a mutation is on hand. This is not at all the mechanism of cross-beta amyloid nor sickle cell hemoglobin aggregation, but again the result is late onset dementia in long-lived non-dividing neurons. Unlike cross-beta sheet, which unrelated protein fragments from many genes can form, loop-sheet polymerization will be limited to serpins.

Aberrant protein processing with tissue deposition is associated with many common neurodegenerative disorders.. Substantial progress has been made toward understanding the pathophysiology of prototypical conformational diseases and protein polymerization in the superfamily of serine proteinase inhibitors (serpins). [See Curr. Opin. Struct. Biol. 8, 799-809 (1998). Lancet 350, 134-138 (1997),

Here we describe a new disease, familial encephalopathy with neuroserpin inclusion bodies (FENIB), characterized clinically as an autosomal dominantly inherited pre-senile dementia, histologically by unique neuronal inclusion bodies and biochemically by polymers of the neuron-specific serpin, neuroserpin. We report the cosegregation of point mutations in the neuroserpin gene with the disease in two families. One mutation, S49P, has homology to a previously described serpin mutation; the other's effect, S52R, is predicted by modelling of the serpin template. Inhibitors of protein polymerization may be effective therapies for this disorder and perhaps for other more common neurodegenerative diseases.

In the larger family, affected individuals have cognitive decline including deficits in attention and concentration, response regulation difficulties and impaired visuospatial skills in their fifties. After several years of disease progression, most affected individuals are unable to work and eventually require nursing-home care. The second smaller family has onset in their thirties with both epilepsy and progressive cognitive decline ending in institutionalization. Eosinophilic neuronal inclusions (called Collins bodies) are distributed throughout the deeper layers of the cerebral cortex and in many subcortical nuclei, especially the substantia nigra . The inclusions are 5-50 µm in diameter and strongly periodic acid-Schiff (PAS) positive, but diastase resistant. They are distinctly different from Lewy bodies, Pick bodies and Lafora bodies. [here is no mention of Congo Red but see alpha 1-antitrypsin serpin study. FENIB is not an amyloidosis and not sickle cell hemoglobin either.]

The inclusions were identified as neuroserpin from amino-acid sequencing. The neuroserpin protein consists of 410 amino acids and is glycosylated [at 3 sites] to a final gene product of molecular weight 55,000 [lacking the 16 aa signal peptide]. The neuroserpin gene (PI12) has been mapped to 3q26 [Genomics 40, 55-62 (1997)]; it has 8 exons. S49P occurs in exon 2. This mutation occurs in 14 individuals out of the 38 family members screened but not in 120 controls in the PI12 Syracuse kindred. [See also: OMIM entry] S52R, called PI12 Portland, is found in both a mother and her older child, but not in two unaffected family members.

[Chicken neuroserpin was the first to be sequenced; it has 80% identity with human. Mouse has 9 exons with boundaries just like thouse of its nearest homologues, protease nexin-1 and plasminogen activator inhibitor-1. The promoter is TATA-less with a CAAT box and several sites for the general transcription factor Sp1 and the neuron-specific transcription factor AP-2 (not unlike the prion gene). -- webmaster]

Neuroserpin is a typical member of the serpins, a family of serine proteinase inhibitors that share a tightly conserved tertiary structure [see PDB entry for human antithrombin-III]. The inhibitory function of the serpins depends on their ability to undergo a profound conformational transition, involving an opening of the main five-stranded beta sheet of the molecule, and allowing the insertion of the reactive centre loop as an extra middle strand. [Two extremely poor quality graphics illustrate this concept.] The control of this sheet-opening mechanism is crucial to the stability of the molecule, and mutations that favour the ready opening of the sheet facilitate pathological intermolecular linkages, with the reactive loop of one molecule inserting into the opened sheet of the next to give loop-sheet polymers. [These certainly are not cross-beta amyloid -- webmaster]

Such loop-sheet polymerization of conformationally unstable mutants in alpha 1-antitrypsin results in inclusion-body formation in hepatocytes. An overall analysis of thse variants shows a clustering of mutations in the domain centred on Ser 49, which when substituted by proline will destabilize the sheet opening mechanism, probably leading to formation of loop-sheet polymers.

Most pertinent to the neuroserpin abnormality is the model provided by the various mutations of 1-antitrypsin resulting in liver disease. These answer the question posed by other neurodegenerative disorders, such as the spongiform encephalopathies and Alzheimer's disease, as to whether protein deposition and accumulation is in itself sufficient to explain the late-onset dementia. The answer from 1-antitrypsin-associated liver disease is yes; hepatocyte loss and eventual cirrhosis is a consequence of variant deposition and not loss of function, as cirrhosis only develops with a conformationally unstable variant and not with those mutations giving complete 'null' suppression of synthesis. [This hardly shows that the polymer causes the disease and offers nothing by way of a pathogenic mechanism. -- webmaster]

The serious consequences of neuroserpin instability are relevant to the dementias as a whole because of the much greater risk that protein accumulation poses to long-lived and non-dividing cells such as neurons; the damage to neurons from such protein deposition will be cumulative and irreversible, in keeping with the observed consistency and late onset of presentation of the dementias in general. FENIB thus conforms to the general pattern of neurodegeneration caused by aberrant protein processing and tissue deposition.

Hereditary cardiac amyloidosis

American Journal of Pathology. 1999;155:695-702
Laura Obici, ...,  and Giampaolo Merlini 
We identified a novel missense mutation in the apolipoprotein A-I gene, T2069C L174 S, in a patient affected by familial systemic nonneuropathic amyloidosis. The amyloid deposits mostly affected the heart of the proband, who underwent transplantation for end-stage congestive heart failure. Amyloid fibrils of myocardial and periumbilical fat samples immunoreacted exclusively with anti-ApoA-I antibodies.

Amyloid fibrils extracted from the heart were constituted, according to amino acid sequencing and mass spectrometry analysis, by an amino-terminal polypeptide ending at Val93 of apolipoprotein A-I (apoA-I); no other significant fragments were detected.

The mutation segregates with the disease; it was demonstrated in the proband and in an affected uncle and excluded in three healthy siblings [suggesting autosomal dominance; see OMIM -- webmaster]. The plasma levels of high-density lipoprotein and apoA-I were significantly lower in the patient than in unaffected individuals.

This represents the first case [not really, R173P was reported in Apr 99 -- webmaster] of familial apoA-I amyloidosis in which the mutation is outside the polypeptide fragment deposited as fibrils. Visualization of the mutation in the three-dimensional structure of lipid-free apoA-I, composed of four identical polypeptide chains, indicates that position 174 of one chain is located near position 93 of an adjacent chain and suggests that the amino acid replacement in position 174 is permissive for a proteolytic split at the C-terminal of Val93.

Comment (webmaster): These are clasical congophilic cross-beta fibrils that end variously in the 83-94 residue amino terminus. Perhaps a half dozen mutants are known; for a time, amyloidosis was erroneously attributed to extra charge. Position 173-4 are of interest because Milano A-1, R173C, is not amyloidogenic. Fragments of protein are involved in many diseases of this type and enhanced proteolysis comes up most notably in Alzheimer.

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