Fly larvae and pupae as vectors for scrapie
In vitro conditions for prion fibril formation.
Transmissible and genetic prion diseases share a common pathway
Germany: 12 generations of "fatal familial insomnia"
Nmr structure of PrP106-126
Upregulation of lysosomal hydrolases, perforin, and peroxidases in TSE mice
Transmission of 3 mouse-adapted scrapie strains to murine neuroblastoma cell lines
Genes on 187,029 bp of flanking sequence of prion-doppel
New familial encephalopathy with neuroserpin inclusion bodies
Copper potentiation of Alzheimer Abeta neurotoxicity
4 December 1999 Lancet 354, Number 9194 Karin Post, Detlev Riesner, Volker Walldorf, Heinz MehlhornBackground (webmaster):
|Infestation by larvae of flies is called myiasis. S.carnaria is among the most common agents of myiasis. After deposition of eggs on skin, larvae of flies can burrow into the skin or penetrate in wounds and subcutaneous tissues where they develop to adults. The clinical pattern depends on the species of fly and on the site of infestation.
Larvae can be found in the skin, in body cavities (nasal, auditory channel and sinuses) and in natural tracts (gastrointestinal, urinary). Skin infestation is the most common form and may present as furuncular (large painful and pruritic nodules, creeping eruptions, or slowly progressive serpiginous and erythematous tracts.
Flesh flies resemble large houseflies, and many are marked with longitudinal stripes on the thorax and abdomen. Most lay eggs, but in a few species the eggs are retained in the abdomen of the female until they hatch. The larvae develop for about a day, then burrow in meat for a week to ten days before entering the two-week pupal stage. Sarcophaga sarraciniae is a familiar pest in America, S. carnaria in Europe.
Article highlights:P> "We analysed experimental transmissibility of the scrapie agent by natural vectors. a fly, Sacrophaga carnaria, fed with brains of scrapie-infected hamsters in different developmental stages caused scrapie in hamsters after they ate fly extracts.
According to the prion model the scrapie agent consists mainly if not entirely of an abnormal isoform (PrPSc) of the cellular prion protein (PrPC). The mode of natural transmission of the infectious agent is unknown, although a scrapie-free flock of sheep developed scrapie after being in contact with a scrapie-positive flock, which led to the hypothesis that there may be a reservoir for scrapie infectivity in fields.
Attempts to transmit scrapie by nematodes has failed, but analysis of hay mites from scrapie-affected farms revealed infected mites. To analyse natural vector systems, larvae of meat-eating and myiasis-causing flies (Sarcophaga carnaria) and grass mites were exposed to scrapie infectious material. They were tested for their potential to accumulate PrPSc as determined by Western blotting and their ability to transmit scrapie in the hamster model.
16 larvae of S carnaria were fed with 2 mg of either brains of scrapie-infected hamsters (scrapie strain 263K) or healthy control brains. Every 2 days, larvae and pupae, respectively, were assayed for the presence of Proteinase K (PK) resistant PrP. Samples of S carnaria were washed, separated from their outer cuticula and homogenised in TBS-buffer. The homogenates were analysed for PrP with and without PK-digestion and western blotting.5 2 days after eating PrPSc positive brains, larvae showed high amounts of PK-resistant PrP whereas in larvae eating healthy control brains no PrP was detectable. Several days later, neither in living larvae having eaten PrPSc nor in the control group was PrP detectable.
However, in infected larvae who died, PK-resistant PrP was detectable after 14 days (data not shown), indicating a high stability of PrP and supposed conservation of infectivity after endogenous metabolism ended. In separate studies, the inner organs of six third instar larvae, fed with PrPSc infected brains for 2 days, were dissolved in 3 ml 0·85% NaCI and given by oesophageal tube to eight hamsters. 10 days after being fed infected brains, six fly pupae were given orally to four hamsters.
In another study, 200 oribatid and other mites were exposed to infected hamster brain in a glass bottle for 10 days; 60 mites were dissolved in 1 mL 0.85% NaCI and given orally to two hamsters. As a control for all transmission studies infected hamster brain was homogenised in 1 mL 0.85% NaCI and given orally to four hamsters. Animals were observed regularly and killed when they showed signs of scrapie for assay for PrPSc by PK-digestion and western blotting. All remaining hamsters were killed 215 days after the start of the experiment and also examined.
The table shows the distribution of infectivity as determined by incubation time, the number of animals with signs of scrapie, and the appearance of PrPSc. Six of eight hamsters, orally inoculated with larvae contents, became ill, but only five of them were PrPSc positive. Two of four pupae-contents-inoculated hamsters developed clinical signs of scrapie, and three were positive for PrPSc.
None of both hamsters inoculated with mites showed clinical signs of scrapie or PrPSc. Two of four hamsters infected with brain material developed scrapie within short incubation times and were PrPSc positive. The irregular distribution of infectivity results probably from incomplete homogenation of the inoculum.
We conclude that the contents of larvae and pupae from flies having eaten infectious hamster brains can transmit scrapie. This strongly suggest a conservation of infectivity in larvae and pupae. Whether they replicate infectivity as postulated by Wisniewski and colleagues is unknown and appears improbable because no PrP-encoding gene in any insect has been reported. [There is certainly no such gene in Drosophila. -- webmaster.]
Our transmission experiments using mites failed. Probably the number of mites inoculated was too low. The findings of the present work suggest that prion diseases might be transmitted by flies in different development stages, even after death. Our results may be relevant also to transmission of bovine spongiform encephalopathy.
Fitzsimmons WM, Pattison IH. Unsuccessful attempts to transmit scrapie by nematode parasites. Res vet Sci 1968; 9: 281-83. Wisniewski HM, Sigurdarson S, Rubenstein R, Kascsak RJ, Carp RI. Mites as vectors for scrapie. Lancet 1996; 347: 1114.
Question: Are really the flies meat-eating, or do only their larvae eat meat?
Answer: It's the larvae that eat meat. (Commentary provided by Dr. MIchael Hansen, Consumers' Union)
Were 16 larvae fed with scrapie brain and 16 larvae fed with normal brain or were there 8 in each group? Did each larvae get 2 mg or 8 or 16? What does scrapie-infected hamsters actually mean? Were they already in the clinical phase or at which day after inection with which dosis? How many larvae or pupae were assayed for how many days? What does samples mean -- whole larvae or which parts of them? How many larvae died and showed PK-resistant PrP? Were these organs from the 16 or 32 larvae or from additional larvae?
Answer: There were 16 S. carnaria in each group fed with 2 mg of either scrapie-infested or healthy control brains. Knowing the number of larvae/pupae assayed is not critical. But it appears that they were assayed for at least 14 days, which is probably the length of time of the larval stage of S. carnaria (14 days seems about right). Samples refer to whole larvae. The scientists only removed the noncellular on of the fly's outer "skin" (the proper zoological term is "cuticula" which is composed of chitin, protein and often other hardening substances) and ground up the inner portions of the larvae which contains all the organs.
It sounds like it was found in all the infected larvae who died, because otherwise the authors would say that "in some infected larvae who died . . ." These clearly were from additional larvae as the text says they were "separate studies." Further, the inner organs of these third instar larvae were dissolved, while the 16 larvae fed brains from either healthy or scrapie-infested hamsters, were homogenized and tested, at various time periods, for the presence of PrPSc. Homogenization is different from dissolving the inner organs in a salt solution. So this is clearly a second experiment.
Question: How is this possible: "10 days after being fed infected brains, six fly pupae were given orally to four hamsters?"6 infected larvae were allowed to develop to pupae and the organs of further 6 larvae were used as third instar for the oral infection of hamsters. In addition every 2 days, larvae and pupae, respectively, were assayed for the presence of Proteinase K (PK) resistant PrP. How many did they use?
Answer: The pupae feeding experiment is yet a third experiment.
Question: What does third instar and oribatid mean?
Answer: This would be a fourth experiment. Oribatid just refers to a family of mites (Oribatidae). Instar refers to stages in insect and mite development between molts (i.e. shedding of the outer "skin"). In many mites, there is an egg stage, then 3 larval instars (the outer skin of insects and mites is hard; when the organism has grown sufficiently, it must shed that "skin" so that it can become larger), followed by a pupal stage (and perhaps a prepupal stage, both of which are "resting" stages), followed by emergence of the adult. So, by using the third instar, they're using the last and largest larval stage.
Question: In all transmission studies infected hamster brain was homogenised in 1 mL 0.85% NaCI and given orally to four hamsters. Was it 1 ml with or without the brain and how many grams of how many hamsters was this brain? Was it 0.85% wheight per volume or volume per volume and was it 0.85% with or without the brain and what is NaCI? Should not all regions of scrapie infected hamster brains be infective and is it really so difficult to homogenize a hamster brain?
Answer: 1 mL refers to a 1 molar solution (ml is the abbreviation for milliliter, mL is the abbreviation for molar) and NaCl refers to sodium chloride (chemical formula is NaCl). Depending on the strain of scrapie, different subportions of the brain may be differentially infective. In fact, there will probably be some subportions of the brain with no detectable PrPSc. Further, there were 3 different sources of the inoculum: S. carnaria larvae, S. carnaria pupae, or hamster brain and so the "incomplete homogenization" would refer to all 3 sources of inoculum as in none of them were all the exposed hamsters infected.
Question: Several days after feeding of PrPSc in living larvae no PrP was detectable. So what does conservation of infectivity in larvae really mean? If there was replication of the infectivity, why wasn't it detectable after several days?
Answer: It means that PrPSc wasn't detected in the living larvae until a number of days after they started eating infective brain. That's not suprising. Even with scrapie, you often can't detect it in hamsters for a few days after the first inoculation happens. However, the fact that the tissue of S. carnaria larvae and pupae, when fed to hamsters caused infection strongly suggests that PrPSc was present in the larvae and pupae.
The authors aren't suggesting that the PrPSc replicates in S. carnaria, simply that the PrPSc hangs around in the fly's body and is present at high enough levels to infect hamsters. This isn't all that surprising either as Race and Chesbro found a similar same thing when they put hamster scrapie into mice, which showed no symptoms of a TSE for over a year, but the spleens and brains of these asymptomatic mice cause hamster scrapie when injected into hamsters (Race and Chesbro. 1998. Scrapie infectivity found in resistant species. Nature 392: 770). This suggested that the hamster PrPSc stayed around in the body of the mice, without causing disease, but was still able to reinfect hamster.
Eur J Biochem 1999 Dec 15;266(3):1192-120 Ragg E, Tagliavini F, Malesani P, Monticelli L, Bugiani O, Forloni G, Salmona MComment (webmaster): This is interesting work though solvent conditions leave something to be desired. Some aspects of the conformation seem quite robust however. The focus on 106-126 has historical justification but the actual protein domain boundary would seem to be 99-127 (which would incidentally help with solubility). The first sentence of the abstract is apparently a typo; they do not mean -128. Recall hamster nmr included residue 112. Neither study presents a definitive native ambient environment.
The main idea proposed for this strongly conserved domain is a hinge region that -- after some copper has been captured to protect the synapse -- brings the condensed repeat region around to the globular domain . As the domain must serve two different masters conformationally speaking, mutational changes that work for both are not feasible, accounting for its extreme invariance despite a bland primary sequence of residues incapable of being in an active site.
PHGGGWGQGGGTHSQWNKPSKP 106-KTNMKHMAGAAAAGAVVGGLGGY-128 MLG
In deionized water at pH 3.5, the peptide adopted a helical conformation in the hydrophobic region spanning residues Met112-Leu125, with the most populated helical region corresponding to the Ala115-Ala119 segment ( approximately 10%). In trifluoroethanol/H2O, the alpha-helix increased in population especially in the Gly119-Val122 tract ( approximately 25%).
The conformation of this region was found to be remarkably sensitive to pH, as the Ala120-Gly124 tract shifted to an extended conformation at pH 7. In dimethylsulfoxide, the hydrophobic cluster adopted a prevalently extended conformation. For all tested solvents the region spanning residues Asn108-Met112 was present in a 'turn-like' conformation and included His111, situated just before the starting point of the alpha-helix.
Rather than by conformational changes, the effect of His111 is exerted by changes in its hydrophobicity, triggering aggregation. The amphiphilic properties and the pH-dependent ionizable side-chain of His111 may thus be important for the modulation of the conformational mobility and heterogeneity of PrP106-126.
Journal of Virology, January 2000, p. 411-417, Vol. 74, No. 1 Juraj Kopacek, Suehiro Sakaguchi,... Masami Niwa, and Shigeru KatamineIn an attempt to identify the molecules involved in the pathogenesis of prion diseases, we performed cDNA subtraction on the brain tissues of mice affected with an experimental prion disease and the unaffected control.
The genes identified as being upregulated in the prion-affected brain tissue included those encoding a series of lysosomal hydrolases (lysozyme M and both isoforms of -N-acetylhexosaminidase), a perforin-like protein (macrophage proliferation-specific gene-1 [MPS-1]), and an oxygen radical scavenger (peroxiredoxin).
Dramatic increases in the expression level occurred at between 12 and 16 weeks after intracerebral inoculation of the prion, coinciding with the onset of spongiform degeneration. The proteinase K-resistant prion protein (PrPSc) became detectable by immunoblotting well before 12 weeks, suggesting a causal relationship between this and the gene activation.
Immunohistochemistry paired with in situ hybridization on sections of the affected brain tissue revealed that expression of the peroxiredoxin gene was detectable only in astrocytes and was noted throughout the affected brain tissue. On the other hand, the genes for the lysosomal hydrolases and MPS-1 were overexpressed exclusively by microglia, which colocalized with the spongiform morphological changes. a crucial role for microglia in the spongiform degeneration by their production of neurotoxic substances, and possibly via the aberrant activation of the lysosomal system, would have to be considered.
Journal of Virology, January 2000, p. 320-325, Vol. 74, No. 1 Noriyuki Nishida, David A. Harris, Didier Vilette,...and Sylvain LehmannPropagation of the agents responsible for transmissible spongiform encephalopathies (TSEs) in cultured cells has been achieved for only a few cell lines. To establish efficient and versatile models for transmission, we developed neuroblastoma cell lines overexpressing type a mouse prion protein, MoPrPC-A, and then tested the susceptibility of the cells to several different mouse-adapted scrapie strains.
The transfected cell clones expressed up to sixfold-higher levels of PrPC than the untransfected cells. Even after 30 passages, we were able to detect an abnormal proteinase K-resistant form of prion protein, PrPSc, in the agent-inoculated PrP-overexpressing cells, while no PrPSc was detectable in the untransfected cells after 3 passages.
Production of PrPSc in these cells was also higher and more stable than that seen in scrapie-infected neuroblastoma cells (ScN2a). The transfected cells were susceptible to PrPSc-A strains Chandler, 139A, and 22L but not to PrPSc-B strains 87V and 22A. We further demonstrate the successful transmission of PrPSc from infected cells to other uninfected cells.
Our results corroborate the hypothesis that the successful transmission of agents ex vivo depends on both expression levels of host PrPC and the sequence of PrPSc. This new ex vivo transmission model will facilitate research into the mechanism of host-agent interactions, such as the species barrier and strain diversity, and provides a basis for the development of highly susceptible cell lines that could be used in diagnostic and therapeutic approaches to the TSEs.
Am J Med Genet 1999 Dec 3;87(4):311-316 Harder A, Jendroska K, Kreuz F, Wirth T,.. Windl O, Kretzschmar HA, Nurnberg P, Witkowski RWe present a novel large German kindred of fatal familial insomnia (FFI) consisting of three branches and comprising more than 800 individuals of 12 generations, the largest pedigree of any familial prion disease known today. There is a wide spectrum of clinical presentations leading to misdiagnoses of Olivo-Ponto-Cerebellar Atrophy (OPCA), Parkinson's or Alzheimer's disease in addition to Creutzfeldt-Jakob disease (CJD) and Gerstmann-Straussler-Scheinker (GSS) syndrome.
Molecular genetic analysis of the prion protein gene (PRNP) confirmed the mutation D178N segregating with methionine at the polymorphic codon 129 of PRNP in all 7 patients examined. This polymorphism at codon 129 is supposed to discriminate between familial CJD (fCJD) and FFI; the 129M allele determines FFI and 129V fCJD.
Furthermore, heterozygosity at this site appears to induce prolonged disease duration as compared to the homozygous condition. The variability of the clinical and pathological findings documented for our patients indicates the difficulty in establishing the diagnosis of FFI on clinical and on pathological grounds alone. In three cases (IX-97, XI-21, V-2) followed up by us prospectively insomnia was an early and severe symptom; however, in case notes analyzed retrospectively this symptom was frequently missed.
In contrast to previous reports and in agreement with recent studies we cannot confirm a clear relationship between the status of the M/V polymorphism at codon 129 and the age-of-onset of this disease.
Comment (webmaster): "FFI" and "GSS" never made the slightest sense as distinct diseases -- there is nothing like looking at a single large kindred to see the overwhelming variation of symptoms that cannot be objectively distinguished from CJD. It is more accurate to simply call it all CJD and give the haplotype (there are tens of millions of these combinatorically, given the mutation set).
This is not the largest known kindred, though it is one of the oldest. The Indiana kindred F198S is said in the literature to comprise 3000 individuals. In a large kindred, variation in presentation might mapped to secondary genes but no one has pursued this in this particular disease; SNPs make this ever more feasible.
No one in prion research has yet bestirred themselves to look at anything but the coding sequence in CJD so other factors could still be nearby. Of the 2,611 bp of exonic prion sequence, 762 bp is coding (29%). In many diseases, half or more of the disease loci map to untranslated regions of the gene.
TSEs are inherently sensitive to upregulation. Downregulation has odd effects as well as seen (mouse doppel ataxia) by knocking out the prion exon 3 splice acceptor. But no one has the energy to look a meagre 30bp upstream of CDS in "sporadic" CJD. (Bizarely, this will be done in chronic wasting disease before in humans.)
Sporadic CJD is thus a misnomer. It should instead be called sporadic-cds CJD, meaning the protein coding sequences were determined to be wild type or neutral alleles. If the CDS and first splice junction checked out, call it sporadic-2:3cds CJD. If no work-up was done, it might better be called unclassified CJD or lazy doctor CJD. It makes increasing sense in neurological disorders to routinely sequence a panel of commonly suspected genes before spending a fortune on other (often invasive) diagnostic tests.
Today one can send out an entire novel bacterial genome for sequencing (two day turn-around) by InCyte Pharmaceuticals, so easily 50 complete prion-doppel regions (41kb) a day. This is far more efficient than dozens of small labs trying their hand at sequencing contigs. And InCyte no doubt would do a better job of it.
Prior to identification, kindreds are also very interesting for the misdiagnoses they evoke, here at death olivoponto cerebellar atrophy, Parkinson, Alzheimer, CJD, and GSS. One wonders how many cases of olivoponto cerebellar atrophy, Parkinson, and Alzheimer are diagnosed each year in Germany (or rather, how many cases are immuno-autospsied each year)?
21 Dec 99. Imagery from webmaster's new gene prediction tool, GeneBanderUsing data from GenBank and the Sanger Center chromosome 20 team, the webmaster assembled a contig of 187,029 consecutive nucleotides on human chromosome 20 centered around the 60,000 bp prion-doppel douplet, consisting of contigs 5'+dJ189G13.00057,-dJ1068H6, [HSJ1187J4] and dJ599I11.
A region of 30,000 bp containing the prion gene and its CpG island, is shown below. GeneBander is capable of annotating a million base pairs a day at this resolution. Chromosome 20 will be completely sequenced in coming months.
RepeatMasker summary: 187029 bp; GC: 43.78 % bases masked 102082 bp (54.58 %) Total interspersed repeats: 99524 bp 53.21 % SINEs: 100 25022 bp 13.38 % ALUs 90 23494 bp 12.56 % MIRs 10 1528 bp 0.82 % LINEs: 71 44452 bp 23.77 % LINE1 47 36846 bp 19.70 % LINE2 19 6144 bp 3.29 % LTR elements: 48 22370 bp 11.96 % MaLRs 15 8974 bp 4.80 % Retrov. 17 6911 bp 3.70 % MER4_group 7 3035 bp 1.62 % DNA elements: 32 7680 bp 4.11 % MER1_type 19 3264 bp 1.75 % MER2_type 7 3278 bp 1.75 % Satellites: 1 261 bp 0.14 % Simple repeats: 25 1448 bp 0.77 % Low complexity: 19 874 bp 0.47 %
Chromosome 22, which is completely sequenced now, had 14,000,000 bp of identifiable trash or 41.9%) Recall that on the whole of chromosome 22, some 629 features were found in 33.4 mbp (1 feature per 53,000 bp, similar to here, 1 in 47,000 bp) by all methods considered together:
247 proteins already known to be on chr 22 150 proteins related to known proteins in human or other species 148 apparent genes (matches only to mRNA ESTs) 545 total functional genes 107 processed retrotransposed mRNA pseudogenes 27 pseudogenes with introns (duplicative genomic transpositions) 134 total pseudogenes (very likely too low)On chr 22, GenScan predicted 6,684 exons in 817 genes of which 325 are not in the above classes; 225 anticipated to be false positives and 100 anticipated to later get database or experimental support. The sensitivity of GenScan is that the 492 it found would be 90% of the functional genes or 78% of the total features.
Gene candidates in the prion-doppel region:
Note: there are two other inherited diseases known to map right on top of the prion-doppel region on chr 20. One is a childhood corneal blindness and the other is a Parkinson-like dementia. While these diseases might not associated with either the prion or doppel gene, finding them is a spin-off of gene discovery in this region.
Three new genes (or gene pieces) did turn up in annotating the 187,000bp contig, all on the 3' end. The region at this point looks like:
5' + psRSP4X + prion + doppel - psIPP + UHSkeratin + dyneinNOS + unknown - 3'
One candidate gene has to do with human dynein light chain 1 cytoplasmic (protein inhibitor of neuronal nitric oxide synthase). At first glance, this seems to be a very recent pseudogene of pure 3'UTR, positions 14617-14696 in AL133396 numbering though more of the gene could be off the end. Another weaker candidate from a single high quality mRNA, apparently has to do with a ultra-high sulfur keratin.
The third is also represented by a single long high quality EST. The best translation of it is a 90 amino acid entity about which nothing is known, QKILHGFKLKIAMLILLKFSFQQCFLAFKHFSNLFKCLQNLTVKSCTHSKLHSVIASLPK IDNTKLLHEICFYKTSQELPAPLAEGY-
Comment (webmaster): The first article is an interesting one as heparan sulfate proteoglycan have a history with CJD too and because of the last sentence. The second discusses 'Collins bodies' which apparently are congophilic neuroserpin fibrils, ie, a new autosomal dominant inclusion body disease. There was no access to full text.
American Journal of Pathology. 1999;155:2115-2125 Marcel M. Verbeek,... and RMW de WaalHeparan sulfate proteoglycans (HSPGs) have been suggested to play an important role in the formation and persistence of senile plaques and neurofibrillary tangles in dementia of the Alzheimer¹s type (DAT). We performed a comparative immunohistochemical analysis of the expression of the HSPGs agrin, perlecan, glypican-1, and syndecans 13 in the lesions of DAT brain neocortex and hippocampus.
Using a panel of specific antibodies directed against the protein backbone of the various HSPG species and against the glycosaminoglycan (GAG) side-chains, we demonstrated the following. The basement membrane-associated HSPG, agrin, is widely expressed in senile plaques, neurofibrillary tangles and cerebral blood vessels, whereas the expression of the other basement membrane-associated HSPG, perlecan, is lacking in senile plaques and neurofibrillary tangles and is restricted to the cerebral vasculature.
Glypican and three different syndecans, all cell membrane-associated HSPG species, are also expressed in senile plaques and neurofibrillary tangles, albeit at a lower frequency than agrin. Heparan sulfate GAG side chains are also associated with both senile plaques and neurofibrillary tangles. Our results suggest that glycosaminoglycan side chains of the HSPGs agrin, syndecan, and glypican, but not perlecan, may play an important role in the formation of both senile plaques and neurofibrillary tangles.
In addition, we speculate that agrin, because it contains nine protease-inhibiting domains, may protect the protein aggregates in senile plaques and neurofibrillary tangles against extracellular proteolytic degradation, leading to the persistence of these deposits.
American Journal of Pathology. 1999;155:1901-1913 Richard L. Davis, George H. Collins,...Daniel A. LawrenceWe report on a new familial neurodegenerative disease with associated dementia that has presented clinically in the fifth decade, in both genders, and in each of several generations of a large family from New York State‹a pattern of inheritance consistent with an autosomal dominant mode of transmission.
a key pathological finding is the presence of neuronal inclusion bodies distributed throughout the gray matter of the cerebral cortex and in certain subcortical nuclei. These inclusions are distinct from any described previously and henceforth are identified as Collins bodies. The Collins bodies can be isolated by simple biochemical procedures and have a surprisingly simple composition; neuroserpin (a serine protease inhibitor) is their predominant component.
An affinity-purified antibody against neuroserpin specifically labels the Collins bodies, confirming their chemical composition. Therefore, we propose a new disease entity‹familial encephalopathy with neuroserpin inclusion bodies (FENIB). The conclusion that FENIB is a previously unrecognized neurodegenerative disease is supported by finding Collins bodies in a small kindred from Oregon with familial dementia who are unrelated to the New York family.
The autosomal dominant inheritance strongly suggests that FENIB is caused by mutations in the neuroserpin gene, resulting in intracellular accumulation of the mutant protein.
Nature: 12/16/99 Ramanujan S. Hegde, Patrick Tremblay, Darlene Groth, Stephen J. Dearmond, Stanley B. Prusiner & Vishwanath R. LingappaComment (webmaster): this is a high quality paper that is too complex to readily summarize. It should be read in full. It is a continuation of earlier studies on the role in disease of protein mis-topology at the endoplasmic reticulum.
Prion diseases can be infectious, sporadic and genetic. The infectious forms of these diseases, including bovine spongiform encephalopathy and Creutzfeldt-Jakob disease, are usually characterized by the accumulation in the brain of the transmissible pathogen, an abnormally folded isoform of the prion protein (PrP) termed PrPSc.
However, certain inherited PrP mutations appear to cause neurodegeneration in the absence of PrPSc, working instead by favoured synthesis of CtmPrP, a transmembrane form of PrP. The relationship between the neurodegeneration seen in transmissible prion diseases involving PrPSc and that associated with CtmPrP has remained unclear.
Here we find that the effectiveness of accumulated PrPSc in causing neurodegenerative disease depends upon the predilection of host-encoded PrP to be made in the CtmPrP form. Furthermore, the time course of PrPSc accumulation in transmissible prion disease is followed closely by increased generation of CtmPrP. Thus, the accumulation of PrPSc appears to modulate in trans the events involved in generating or metabolising CtmPrP. Together, these data suggest that the events of CtmPrP-mediated neurodegeneration may represent a common step in the pathogenesis of genetic and infectious prion diseases.
Certain mutations in PrP including A117V alter its biogenesis at the endoplasmic reticulum, causing a higher percentage of PrP molecules to be synthesized in a transmembrane form that we termed CtmPrP (Science 279, 827-834 (1998)). Expression of CtmPrP-favouring mutations in transgenic mice resulted in neurodegenerative changes similar to those observed in prion disease....We generated mice with mutant PrP transgenes differing in their propensity to form CtmPrP and subsequently assessed their susceptibility to PrPSc-induced neurodegeneration.
In vitro translocation products of four mutants of Syrian hamster prion protein alter the amount of CtmPrP synthesized at the endoplasmic reticulum: increased synthesis of the CtmPrP form of PrP is associated with the development of neurodegenerative diseases. More remarkable is the apparent dose response relationship, seen in two ways, between CtmPrP and disease. First, the more strongly CtmPrP synthesis is favoured at the endoplasmic reticulum (KH II > N108l > A117V), the earlier is the onset of spontaneous disease . Second, lowering the level of expression of each of these mutations below an apparent threshold stops both the generation of CtmPrP (Fig. 1d and ref. 9) and development of disease (Table 1). Furthermore, the three transgenic lines expressing the KH II mutation develop disease at times inversely correlated with their respective levels of expression (Table 1). These observations demonstrate that both the CtmPrP-favouring quality of a mutation and its level of expression contribute to the development of neurodegeneration.
The transgenic mice with differing propensities to make CtmPrP were used to establish the relationship between CtmPrP and PrPSc. We first examined the susceptibility to PrPSc of transgenic mice with similar levels of transgene expression but differing propensities to make CtmPrP: these mice were Tg[SHaPrP(STE)] and Tg[SHaPrP(A117V)H]. Upon inoculation with Sc237 hamster prions, we found that the Tg[SHaPrP(STE)] and Tg[SHaPrP(A117V)H] mice developed illness at approximately 323 and 54 days, respectively (Fig. 2a, Table 1). Biochemical analysis of representative mice at the time of disease onset revealed that the Tg[SHaPrP(STE)] mice contained substantially more PrPSc than the Tg[SHaPrP(A117V)H] mice (Fig. 2b). Thus, the transgenic line that generates higher CtmPrP is more susceptible to PrPSc, developing disease at a lower level of overall PrPSc accumulation.
... The data in Fig. 2 demonstrate two important points. First, very different levels of PrPSc accumulation are observed at the time of onset of neurologic dysfunction upon inoculation of the various lines of transgenic mice. Given that the strain of the mice is identical in each case, with the only differences being in the nature and level of expression of the PrP transgene, we conclude that accumulation of protease-resistant PrPSc is not likely to be the most proximate cause of disease; subsequent events (apparently involving CtmPrP) are likely to be involved. Second, there is a relationship between the amount of accumulated PrPSc and the factors that modulate CtmPrP generation. Such a relationship argues that CtmPrP and PrPSc are part of a pathway in which each can potentially influence, either directly or indirectly, the metabolism of the other.
One way to explain the data in Fig. 2g is if accumulation of PrPSc causes increased generation of CtmPrP, which then elicits neurodegeneration. Thus, transgenic mice that have a greater propensity to generate CtmPrP (that is, a high Ctm-index) would require less PrPSc accumulation before CtmPrP generation is increased beyond the threshold needed to produce detectable neurologic disease. This model would explain the inverse relationship observed in Fig. 2g, and makes two additional predictions. First, as transgenic mice that substantially favour synthesis of PrP in the CtmPrP form can entirely circumvent the requirement for PrPSc in the development of neurodegenerative disease, tissue from such mice should not be infectious. Second, CtmPrP levels should increase during the course of PrPSc accumulation in infectious prion disease. These predictions were tested.
To assess the transmissibility of CtmPrP-associated disease, brain homogenate from clinically ill Tg[SHaPrP(KH II)H] mice was inoculated intracerebrally into four hosts: Tg[SHaPrP(KH II)L] mice expressing the KH II mutation at low levels; Tg[SHaPrP] mice overexpressing wild-type SHaPrP; FVB/Prnp0/0 mice with a homozygous disruption of the PrP gene; and Syrian hamsters. As shown in Fig. 3, homogenate from terminally sick Tg[SHaPrP(KH II)H] mice did not induce neurological illness at rates that were different from those inoculated with control Tg[SHaPrP] homogenate when directly compared in three independent hosts. Furthermore, biochemical and pathological examination of representative brain tissue from animals depicted in Fig. 3 at up to 625 days after inoculation did not show any evidence of PrPSc or neurologic disease in either experimental or control animals (data not shown).
However, inoculation of Tg[SHaPrP(KH II)L] mice with Sc237 prions readily generated PrPSc in brain tissue (Fig. 2d), which re-transmitted disease to Tg[SHaPrP(KH II)L] and Tg[SHaPrP] mice (data not shown). Although PrP(KH II) is capable of being converted into PrPSc, the CtmPrP-associated disease in Tg[SHaPrP(KH II)H] mice does not generate detectable PrPSc and is therefore not infectious. Lack of transmission provides further support for the hypothesis that neurodegeneration in these genetic prion diseases is caused by CtmPrP directly.
The second prediction made on the basis of the data in Fig. 2g was that accumulation of PrPSc in infectious prion disease should induce increased generation of CtmPrP, which would subsequently lead to neurodegeneration. Unfortunately, testing this prediction directly is hampered by the biochemical properties of the accumulating PrPSc (ref. 11). Being highly protease-resistant and heterogeneous in its fractionation, it tends to contaminate substantially all subcellular fractions. In addition, it interferes with the assays for CtmPrP detection, which are also based on protection from proteases. Because PrPSc is not readily degraded by the cell and accumulates to very high levels12, even very small amounts of contamination of subcellular fractions are sufficient to make detection of slight increases in CtmPrP difficult. Thus, an alternate method is required to monitor the effect of accumulating PrPSc on the topology of newly synthesized PrP (see Fig. 4a). (114k)
To design such an experiment, we took advantage of three observations. First, a species barrier to PrPSc conversion exists between mouse and Syrian hamster13,14. Second, in contrast to the species barrier for PrPSc formation, no species-specific differences in the synthesis, translocation or topology are observed between mouse prion protein (MoPrP) and SHaPrP (ref. 9 and data not shown). And, finally, monoclonal antibodies highly specific to SHaPrP (which do not cross-react with MoPrP) are available to distinguish between expression of these two PrP transgenes15. Thus, in such double-transgenic animals we can use hamster-CtmPrP formation as a 'reporter' of signalling in trans during the course of accumulation of mouse PrPSc. For this experiment, the double-transgenic mice which synthesize both MoPrP and SHaPrP (see Methods) are inoculated with mouse prions (of the RML strain). Then, at various intervals during the time course of accumulation of PrPSc and development of disease, individual mice are killed and examined for total PrPSc accumulation and for the presence of hamster CtmPrP (see Fig. 4a). The principle is that, following inoculation, only MoPrP will be a substrate for prion replication and PrPSc formation13. The effect of this PrPSc accumulation on the ability of cells to generate (or not generate) CtmPrP can be assessed by examining SHaPrP.
Clinical disease developed in these animals about 9 weeks after inoculation (data not shown). We found that PrPSc accumulated in these mice during this 9-week time course, with the earliest times at which it was detectable being approximately 5-6 weeks (Fig. 4b). As expected, the SHaPrP was not found to have formed any PrPSc by both biochemical criteria in this study (Fig. 4b) and infectivity criteria in previous studies13. However, a significant increase in the amount of CtmPrP was noted upon examination of the SHaPrP (Fig. 4c). Such an increase was not observed in a parallel set of mice that did not receive the inoculum (data not shown). These findings, coupled with the observation that CtmPrP can cause neurodegeneration in the absence of a transmissible forms of PrP (ref. 9, Figs 1 and 3), suggest that PrPSc accumulation may cause disease by inducing the synthesis of CtmPrP de novo.
These findings suggest causal relationships between PrPSc accumulation, the events of CtmPrP formation and metabolism, and the development of neurodegenerative disease. Three complementary and independent lines of evidence argue for this conclusion. First, increasing the generation of CtmPrP beyond a certain threshold (by modulating a combination of PrP mutation and level of expression) results in neurodegeneration in the absence of PrPSc formation (Figs 1 and 3 and ref. 9). Second, the amount of accumulated PrPSc needed to cause neurodegenerative disease is influenced by the propensity of the host to generate CtmPrP (Fig. 2). And third, the brain appears to contain increasing levels of CtmPrP during the course of accumulation of PrPSc (Fig. 4). Taken together, the data are suggestive of three successive stages in the pathogenesis of prion diseases (Fig. 5).
Infectious prion diseases are proposed to work by initiating the steps of Stage I, the accumulation of PrPSc. Genetic prion diseases could in principle work at either Stage I or II. If the PrP mutation in question results in the spontaneous formation of PrPSc, Stage I would be initiated, PrPSc would replicate and accumulate, and subsequently cause increased elevation of CtmPrP (Stage II). Such a mechanism seems plausible for the E200K mutation which causes familial Creutzfeldt-Jakob disease16. Thus, PrPSc is seen in these patients17, and the disease is readily transmissible to experimental animals7. Alternatively, certain other PrP mutations could bypass Stage I altogether by directly causing an increase in CtmPrP generation. The A117V mutation resulting in human Gerstmann-Sträussler-Scheinker18 disease seems likely to work by such a mechanism. This would explain why this disease has not been transmissible6,7,8, and why PrPSc has not been detected in the brain tissue of these patients6,9.
The final stage in prion disease pathogenesis includes the mechanisms by which CtmPrP, once generated, leads to neurodegenerative disease. The mechanism by which this occurs and the intracellular pathways that are involved remain entirely unclear. However, it does not appear to be the case that CtmPrP is simply misfolded, retained or accumulated in the endoplasmic reticulum, or that it elicits an unfolded protein response. This is suggested by the observation that essentially all of the CtmPrP has been trafficked beyond the endoplasmic reticulum (ref. 9), which is the site of the presently known quality-control machinery for protein folding in the secretory pathway19,20. In addition, disease can be elicited by transgenes expressed at close to physiologic levels, as is the case with Tg[SHaPrP(KH II)M] animals or human cases of Gerstmann-Sträussler-Scheinker disease containing the A117V mutation. Thus, a more selective means by which CtmPrP induces neurodegeneration is suggested by the available data.
Biochemistry (in press) Swietnicki W, Petersen RB, Gambetti P, Surewicz WKComment (webmaster): As ever better control over the prion protein and its assembly to a fibril are obtained, the day is not far off when infectivity might be established using in vitro fibrils from homogeneous recombinant protein. It may or may not be a good idea to direct therapeutic efforts at the assembly process, depending on whether disassembly is promoted.
A consistent effect of lowered pH is seen in several recent experiments including this one. The fibrils in this paper were thioflavine-positive and 150 angstroms in diameter, therefore amyloid-like. This, in conjunction with a gain in protease K resistance, is suggestive of in vivo relevence (though covalent modifications are lacking).
The GdnHCl/acidic pH-induced transition of huPrP90-231 to a beta-sheet-rich structure is consistently accompanied by protein self-association into large molecular weight aggregates. The transition is protein-concentration dependent, so the conformation shift probably occurs simultaneously with oligomerization; previously proposed intermediate states are probably low oligomerization states, ie, a stable monomeric seed crystal is problematic.
The authors find, like Caughey's lab, that the choice of salt is just as important as the choice of denaturant. Here urea alone was insufficient for aggregation but NaCl + urea aggregated readily. The results here differ importantly from Jackson et al. [(1999) Science 283, 1935-1937.] in that disulphide reduction was not necessary. Turk et al. reported long ago [(1988) Eur. J. Biochem. 176,21-30] that authentic rogue conformer has intact disulfide. This makes it very unlikely that all of the peptide is involved in beta-sheet, consistent with other amyloids.
"According to the ³protein-only² hypothesis, the critical step in the pathogenesis of prion diseases is the conformational transition between the normal and pathological isoforms of prion protein. To gain insight into the mechanism of this transition, we have characterized the biophysical properties of the recombinant protein corresponding to residues 90-231 of the human prion protein (huPrP90-231).
Incubation of the protein under acidic conditions (pH 3.6-5) in the presence of 1 M guanidine-HCl resulted in a time-dependent transition from an alpha-helical conformation to a beta-sheet structure and oligomerization of huPrP90-231 into large molecular weight aggregates. No stable monomeric beta-sheet-rich folding intermediate of the protein could be detected in the present experiments.
Kinetic analysis of the data indicates that the formation of beta-sheet structure and protein oligomerization likely occur concomitantly. The beta-sheet-rich oligomers were characterized by a markedly increased resistance to proteinase K digestion and a fibrillar morphology (i.e., they had the essential physicochemical properties of PrP Sc ).
Contrary to previous suggestions, the conversion of the recombinant prion protein into a PrP Sc -like form could be accomplished under nonreducing conditions, without the need to disrupt the disulfide bond. Experiments in urea indicate that, in addition to acidic pH, another critical factor controlling the transition of huPrP90-231 to an oligomeric beta-sheet structure is the presence of salt. "
J Biol Chem, Vol. 274, Issue 52, 36859-36865, December 24, 1999 Manuel Morillas, Wieslaw Swietnicki, Pierluigi Gambetti, and Witold K. SurewiczThe prion protein (PrP) in a living cell is associated with cellular membranes. However, all previous biophysical studies with the recombinant prion protein have been performed in an aqueous solution. To determine the effect of a membrane environment on the conformational structure of PrP, we studied the interaction of the recombinant human prion protein with model lipid membranes.
The protein was found to bind to acidic lipid-containing membrane vesicles. This interaction is pH-dependent and becomes particularly strong under acidic conditions. Spectroscopic data show that membrane binding of PrP results in a significant ordering of the N-terminal part of the molecule. The folded C-terminal domain, on the other hand, becomes destabilized upon binding to the membrane surface, especially at low pH.
Overall, these results show that the conformational structure and stability of the recombinant human PrP in a membrane environment are substantially different from those of the free protein in solution. These observations have important implications for understanding the mechanism of the conversion between the normal and pathogenic ) forms of prion protein.
J Biol Chem, Vol. 274, Issue 52, 37111-37116, December 24, 1999 Xudong Huangab, Math P. Cuajungcoa,... Rudolph E. Tanzig, and Ashley I. BushakComment (webmaster): There is a potential for two distinct phenomenon involving copper and amyloidoses. One, described below, might be an inherent association of copper with cross-beta secondary structure. This would lead to a universal mechanism of toxicity of the amyloid, irregardless of the originating gene. While this would be applicable to the prion gene as well, its separate ability to bind copper to the repeat region is an opportunity for confusion: the repeat region is generally cleaved off mature prion amyloid, and so would not be involved in copper toxicity of the amyloid.
"Oxidative stress markers as well as high concentrations of copper are found in the vicinity of a amyloid deposits in Alzheimer's disease. The neurotoxicity of Abeta in cell culture has been linked to H2O2 generation by an unknown mechanism. We now report that Cu(II) markedly potentiates the neurotoxicity exhibited by a in cell culture.
The potentiation of toxicity is greatest for A1-42 relative to A1-40 mouse/rat A1-40, corresponding to their relative capacities to reduce Cu(II) to Cu(I), form H2O2 in cell-free assays and to exhibit amyloid pathology. The copper complex of A1-42 has a highly positive formal reduction potential (+500-550 mV versus Ag/AgCl) characteristic of strongly reducing cuproproteins. These findings suggest that certain redox active metal ions may be important in exacerbating and perhaps facilitating Abeta-mediated oxidative damage in Alzheimer's disease."