Prion Deletion and Insertion Mutants
Mad Cow Home or Best Links

Sept 24 CJD in blood meeting agenda
Briefly Noted
New prion structural studies
Alleles found in rogue conformer
New prion analog found
How do prion proteins get to the brain?
Scientists pinpoint role of Alzheimer's genes
Human rabies called CJD in Montana and Washington
Harmless polymorphism?

Alleles found in rogue conformer

The article below addresses one of my pet peeves, that in familial CJD, which is almost always heterozygous, researchers have generally failed to determin which allele products end up as rogue prion and with what N- and C-termini. Two different situations are reported here: D178N is the only mutant allele in protease-resistant product irrespective of codon 129, whereas an long repeat insertion and wild type appeared in another form of the disease. That leaves a dozen or so situations where the composition is still unknown.

The same issue of Nature Medicine has two relevent Alzheimer notes: gamma-secretase is identified as the final protease creating the amyloidogenic peptide, the ragged ends are the Alzheimer 'strain types' with 1-42 the most amyloidogenic.

In the new Nature genetics, the gene for spinocerebellar ataxia, the eighth neurological CAG repeat disorder, has been sequenced, along with a paternal effect. The accession number is not given in the abstract.-- webmaster

Allelic origin of the abnormal prion protein isoform in familial prion diseases

Nature Medicine Vol.3 #9 - September 1997 
 Shu G. Chen, Piero Parchi, Paul Brown, ... & Pierluigi Gambetti
The hallmark of prion diseases is the presence of an aberrant isoform of the prion protein (PrPres) that is insoluble in nondenaturing detergents and resistant to proteases. We investigated the allelic origin of PrPres in brains of subjects heterozygous for the D178N mutation linked to fatal familial insomnia (FFI) and a subtype of Creutzfeldt-Jakob disease (CJD178), as well as for insertional mutations associated with another CJD subtype. We found that in FFI and CJD178 subjects, only mutant PrP was detergent-insoluble and protease-resistant.

Therefore, PrPres derives exclusively from the mutant allele carrying the D178N mutation. In contrast, in the CJD subtype harboring insertional mutations, wild-type PrP was also detergent-insoluble and likely to be protease-resistant. Our findings indicate that the participation of the wild-type PrP in the formation of PrPres depends on the type of mutations, providing an insight into the molecular mechanisms underlying the phenotypic heterogeneity in familial prion diseases.

Distinct sites of intracellular production for Alzheimer's disease Abeta40/42 amyloid peptides

Nature Medicine             Volume 3 Number 9 - September 1997 
Tobias Hartmann1, Sophie C. Bieger2, ... Klaus Unsicker3 & Konrad Beyreuther
The Alzheimer amyloid precursor protein (APP) is cleaved by several proteases, the most studied, but still unidentified ones, are those involved in the release of a fragment of APP, the amyloidogenic beta-protein Abeta. Proteolysis by gamma-secretase is the last processing step resulting in release of Abeta. Cleavage occurs after residue 40 of Abeta [Abeta(1-40)], occasionally after residue 42 [Abeta(1-42)]. Even slightly increased amounts of this Abeta(1-42) might be sufficient to cause Alzheimer's disease (AD) (reviewed in ref. 1, 2). It is thus generally believed that inhibition of this enzyme could aid in prevention of AD.

Cloning of the SCA7 gene reveals a highly unstable CAG repeat expansion

Nature Genetics Volume 17 Number 1 - September 1997 
Gilles David, Nacer Abbas, ... & Alexis Brice 
The gene for spinocerebellar ataxia 7 (SCA7) has been mapped to chromosome 3p12-13. By positional cloning, we have identified a new gene of unknown function containing a CAG repeat that is expanded in SCA7 patients. On mutated alleles, CAG repeat size is highly variable, ranging from 38 to 130 repeats, whereas on normal alleles it ranges from 7 to 17 repeats. Gonadal instability in SCA7 is greater than that observed in any of the seven known neuro-degenerative diseases caused by translated CAG repeat expansions, and is markedly associated with paternal transmissions. SCA7 is the first such disorder in which the degenerative process also affects the retina.

CJD and Blood Transfusion

NIH Bethesda MD Building 31, C Wing, conference room #7  24 Sept 97
NHLBI Paul McCurdy
FDA Jay Epstein
Industry Jason Bablak
Transmission Paul Brown, NINDS
Inactivation Methods Bob Rohwer, Mol Neurovirology, Baltimore
Infectivity Laura Manuelidis, Yale
Immunodiagnosis Richard Kascsak, Staten Island
Transgenic Animal Testing Jiri Sarar /Prusiner lab
Epidemiology Larry Schoenberger, CDC
CJD Lookback Marian Sullivan, Am.Assoc.Blood Banks

Scientists pinpoint role of Alzheimer's genes

By GENE EMERY, Reuters September 4, 1997 
BOSTON A defect in the ability of a cell to properly parcel out its genetic material when it replicates itself may be an underlying cause of most inherited cases of Alzheimer's disease, according to a new study. Five Harvard University researchers report in Friday's issue of the journal "Cell" that they have uncovered the tasks performed by presenilin 1 and presenilin 2. Those genes, which have been implicated in the familial form of Alzheimer's, help govern a cell's ability to make copies of its genetic material and send a complete copy to opposite ends of the cell just before it splits in two.

Alzheimer's, a brain disease that can lead to confusion, memory loss and speech problems, often starts in later middle life and is estimated to afflict more than 10 percent of those over 65 years of age. When presenilin 1 or presenilin 2 is defective, the genetic material is not divvied up properly and the two cells created from the original may not be able to function normally.

The same inability to correctly divide the genetic material is responsible for Down's syndrome, where an extra copy of one of the body's chromosomes produces a distinctive pattern of birth defects, including varying degrees of retardation. Huntington Potter, leader of the Harvard team, said he has long suspected a link between Down's and Alzheimer's because people with Down's syndrome always develop Alzheimer's when they are in their 30s or 40s.

The same chromosome, number 21, responsible for Down's syndrome also carries the genetic code for a telltale protein seen in the brain of Alzheimer's patients. Down's syndrome, which occurs in about 1 in 600 to 650 live births, is marked by mental retardation and physical defects. The evidence for Down's syndrome shows up in nearly every cell in the form of a third, unwanted copy of chromosome 21. Potter said the new research supports the idea that people without Down's syndrome who inherit Alzheimer's disease may developed the extra chromosome in some of their cells but not others because the defect appears later in the person's development.

"Instead of occurring in every cell of the body because the chromosome segregation went wrong during the development of the egg, it occurred during the growth of the individual, so only a small percentage of cells had three copies of the chromosome," Potter said. "This would then cause Alzheimer's disease, but at a later stage because of the fact that you only have a few cells in the body that have this genetic defect."
In a separate preliminary study, Potter and Lisa Geller of Harvard have found that the number of skin cells with an extra copy of chromosome 21 is three times higher in people with Alzheimer's disease than it is in healthy individuals of the same age.

Cell September 5, 1997; 90 (5) 917-27

The function of the presenilins seems to have been found. It seems that they are on the nuclear membrane and involved in chromosome separation during mitosis. Somatic or familial mutation causes trisomy mosaicism, and then over-production results of the main Alzheimer amyloid protein due to a simple gene dosage effect. Down's syndrome, which is chromosome 21 trisomy, is very similar (and invariably results in early Alzheimer's); APP is on chromosome 21.

In other words, the presenilins have nothing particular to do with nervous system functioning. Their effect is over-production of a protein with a propensity to form amyloid. This is similar to multi-gene mouse models of CJD and possibly to T183A and other mutants that may have their main effect through over-production via a mass action effect. -- webmaster

Mutations in two related genes, presenilin 1 and 2, account for most early-onset familial Alzheimer's disease. Although structural features indicate that the presenilins are membrane proteins, their function is unknown. We have localized the presenilins to the nuclear membrane, its associated interphase kinetochores, and the centrosomesall subcellular structures involved in cell cycle regulation and mitosis. The colocalization of the presenilins with kinetochores on the nucleoplasmic surface of the inner nuclear membrane, together with other results, suggests that they may play a role in chromosome organization and segregation, perhaps as kinetochore binding proteins/ receptors. We discuss a pathogenic pathway for familial Alzheimer's disease in which defective presenilin function causes chromosome missegregation during mitosis, resulting in apoptosis and/or trisomy 21 mosaicism.

Alzheimer's disease arises when neurons in certain regions of the brain, particularly those involved in memory and cognition, are damaged and ultimately killed. A key step in this process is the polymerization of the Aş peptide into neurotoxic protein filaments. These filaments accumulate in the brain as the characteristic neuropathological lesions termed "amyloid".

The products of five genes have been implicated in the pathogenic pathway that leads to Alzheimer's disease (for reviews, see Ashall and Goate, 1994 ; Schellenberg, 1995 ). The most prominent is the amyloid precursor protein (APP), which is encoded on chromosome 21 and gives rise to the Aş peptide by proteolytic cleavage (for review, see Selkoe, 1994 ). Rare mutations in the APP gene cause some forms of familial Alzheimer's disease (FAD) by altering APP processing to increase either the total production of Aş or the relative amount of its most amyloidogenic form (Aş1-42). Similarly, all individuals who carry three copies of chromosome 21 (and therefore of the APP gene) due to meiotic nondisjunction (Down syndrome) also produce more Aş1-42 (Teller et al., 1996 ) and develop Alzheimer pathology at an early age (reviewed in Potter, 1991 ). In addition to APP mutations, inheritance of certain alleles of apolipoprotein E (apoE4) and possibly antichymotrypsin (ACT-A) increases the risk of developing Alzheimer's disease. ApoE4 and ACT apparently act by promoting the formation of the neurotoxic amyloid filaments

The majority of early-onset cases of FAD is caused by mutations in either of two related genes, termed the presenilins). Although some 30 point mutations that cause FAD have been identified in PS1 and -2, they have provided little insight into presenilin pathophysiology (for review, see Haass, 1996 ). Thus, while the Aş protein evidently forms the bulk of the amyloid deposits, and apoE4 and ACT have been shown to bind to Aş and promote its polymerization into neurotoxic filaments, the function(s) of the presenilin proteins is unknown.

The first model for presenilin function sought to link known features of Alzheimer's disease with the overall structure of the presenilin proteins. Specifically, the six to eight apparent transmembrane domains in the presenilins and the fact that the two cleavage sites used to generate Aş lie within and adjacent to the membrane-spanning domain of APP suggested to Sherrington et al., 1995 that PS1 and -2 might reside near APP in the plasma membrane or the endoplasmic reticulum where their mutations could alter normal APP processing. The hydrophobic domains in PS1 and PS2 have also prompted hypotheses that the presenilins may reside in the plasma membrane and there function as ion channels, cell adhesion molecules, or G protein-coupled receptors.

We have obtained immunocytochemical evidence that, in normal dividing cells, the Alzheimer presenilin proteins are primarily located in the nuclear membrane, in the centrosomes, and in spots on the inner nuclear membrane associated with interphase kinetochores. These results have several potential implications for the physiological and pathological functions of the presenilins.

The most straightforward interpretation of the results is that presenilin function is related to chromosome organization and segregation and, potentially, to mitosis-specific gene expression. More specifically, their nuclear membrane and kinetochore localization suggests that one function of these proteins may be to serve as kinetochore binding proteins or receptors ("kinetoceptors") to anchor and to organize the chromosomes on the inner nuclear membrane during interphase. In this role, the presenilins might bind DNA directly with special affinity for centromeric sequences, or they might interact with kinetochore proteins, either directly or possibly through microtubules. Indeed, in the likely orientation of the presenilins in the nuclear membrane (Figure 2), the two largest (and most diverse) hydrophilic regions of the two proteins (the N terminus and loop 6) would be oriented toward the nucleoplasm where they could interact with kinetochores and other intranuclear structures and proteins.

In order for a cell to leave G1 and enter mitosis, the kinetochores and their associated chromosomes must be released from the surface of the inner nuclear membrane and its associated proteins such as the presenilins. Two possible ways in which the putative presenilin binding to centromeres/kinetochores might be regulated during this process are by phosphorylation and/or by proteolytic cleavage. It is therefore interesting that the N-terminal, and possibly the loop 6, regions of the presenilins, which face the nucleoplasm, are phosphorylated in vivo by casein kinase 2 and in vitro by the cdc-2 kinase and that both presenilin proteins, though found in full-length form, are also cleaved into two parts in normal and transfected cells.

In addition to chromosome segregation, mitosis is also characterized by changes in gene expression. An indication that the presenilins may be involved in gene expression is provided by the finding that they function in the Notch signaling pathway in C. elegans (Levitan and Greenwald, 1995 ) that controls the asymmetric character of cell division and the differential expression of specific genes during development. Since mutations in LIN-12 (C. elegans Notch) change the pattern of cell division during development, it seems possible, in the light of the results described here, that the regulatory activity of SEL-12 on LIN-12 may involve alterations in chromosome packaging and segregation during mitosis. In addition, the proteolytic cleavage of the presenilin proteins into two parts is reminiscent of the fact that Notch function requires a similar cleavage that releases its C terminus and allows it to translocate to the nucleus).

The localization of the presenilins to intracellular membranes suggests a number of mechanisms by which mutant forms of the proteins could adversely affect cellular physiology and cause Alzheimer's disease. An early proposal was that the presence of the presenilin proteins in the endoplasmic reticulum during their synthesis would place them in contact with the APP gene product in the same subcellular location (Sherrington et al., 1995 ). Here mutant presenilin proteins could affect APP processing and lead to the increased levels of Aş1-42 that has been observed in presenilin mutant individuals, cell lines, and transgenic mice. Consistent with this possibility is the finding that PS1 and PS2 antibodies strongly immunolabel the cytoplasm of neurons in brain sections and in culture. Perhaps in neurons, the presenilins have a dual function involving both cytoplasmic and nuclear membranes.

Another hypothesis for presenilin pathophysiology, which we favor, is based on our finding that the presenilins are associated with three subcellular structures that are all involved in chromatin organization, the nuclear membrane, interphase kinetochores, and centrosome. Specifically, we suggest that the point mutations in the presenilin genes that cause FAD may affect the ability of the presenilin proteins to link the chromosomes to the nuclear membrane and to release them at the appropriate time during mitosis, thus leading to chromosome missegregation and consequent abnormalities such as inappropriate apoptosis.

How could chromosome missegregation lead to Alzheimer's disease? It has been known for some time that individuals with trisomy 21 (Down syndrome) invariably develop Alzheimer neuropathology by age 30-40. This fact implies that three copies of chromosome 21 are capable of causing Alzheimer's disease, possibly through the unbalanced expression of a gene or genes, such as APP, on chromosome 21. In considering the possible relationships between Down syndrome, trisomy 21, and Alzheimer's disease, we developed a hypothesis that suggested that both the genetic and sporadic forms of Alzheimer's may arise from the progressive accumulation of cells with three full copies of chromosome 21 during the life of the individual (Potter, 1991 ). Such trisomy 21 mosaicism, generated by aberrant chromosome segregation during mitosis, could lead to Alzheimer's disease through the same mechanism by which Down syndrome patients develop the disease, but at a later age owing to the modulating effect of the majority population of normal diploid cells.

Several potential mechanisms could explain how trisomy 21 or other aneuploid cells generated by FAD-mutant presenilin proteins could lead to Alzheimer's disease. For example, aneuploid cells might be prone to apoptosis. Indeed, cortical neurons from Down syndrome fetuses undergo spontaneous apoptosis in vitro. Such apoptosis could lead to neurodegeneration directly, but it could also (indirectly) affect APP processing and the production of the Aş peptide. Support for this latter possibility is provided by the findings that apoptosis-prone Down syndrome fetal brains contain a higher ratio of the pathogenic Aş1-42 compared to Aş1-40 and that inducing apoptosis in normal human neurons by serum starvation increases their secretion of the Aş peptide (LeBlanc, 1995 ). A direct connection between the presenilin genes and apoptosis is indicated by the finding that the overexpression of even normal presenilin genes in transfected cells (which we might expect to disrupt orderly chromosome segregation) results in increased sensitivity to apoptotic stimuli. Furthermore, expression of a C-terminal fragment of PS2 in cells was shown to inhibit induced apoptosis, which was then restored by overexpression of full-length PS2 ).

In addition to stimulating apoptosis, aneuploidy (in particular, trisomy 21) promotes inflammation in the braina necessary component of the Alzheimer pathogenic pathway that leads to neurotoxicity (Potter et al., 1995 ). Such inflammation and its acute phase response characterize both Down syndrome and Alzheimer's disease brains and evidently arise from the activation of astrocytes by IL-1 released from hyperactive microglial cells. Unlike neurons, astrocytes and microglia still undergo cell division in the adult brain and therefore would be particularly sensitive to chromosome missegregation induced by mutant presenilin genes.

In sum, the data of this paper indicate that a normal physiological function of the Alzheimer presenilin proteins may be related to mitosis and chromosome organization/segregation. In this role, the presenilins may bind DNA directly, or they may serve as "kinetoceptors," linking the chromosome kinetochores to the inner nuclear membrane. Mutations in the presenilin genes may cause Alzheimer's disease by effecting subtle changes in the function of the proteins that result, for example, in aberrant chromosome segregation. This, in turn, could lead to trisomy 21 mosaicism, apoptosis, or altered APP processing, all of which have been observed in presenilin mutant cells. Gene expression may also be affected, potentially leading to increased production of Alzheimer-related proteins such as APP, ACT, or apoE.

Cell surface expression of the Alzheimer disease-related presenilinİproteins

PNAS Vol. 94, pp. 9926-9931, September 1997  
Nazneen N. Dewji*, and S. J. Singer
The presenilin proteins PS-1 and PS-2 are crucially involved in Alzheimer disease (AD), but their molecular functions are not known. They are integral membrane proteins, but whether they can be expressed at the surface of cells has been in dispute. Here we show by immunofluorescence experiments, using anti-peptide antibodies specific for either PS-1 or PS-2, that live cultured DAMI cells and differentiated human NT2N neuronal cells are specifically immunolabeled for their endogenous as well as transfected presenilins, although the cells cannot be immunolabeled for their intracellular tubulin, unless they are first fixed and permeabilized. These and other results establish that portions of the presenilins are indeed expressed at the surfaces of these cells. These findings support our previous proposal that the presenilins on the surface of a cell engage in intercellular interactions with the -amyloid precursor protein on the surface of a neighboring cell, as a critical step in the molecular and cellular mechanisms that lead to AD.

How do prion proteins get to the brain?

Jane Bradbury Lancet 6 Sept 97 pg839 New data explaining how abnormal prion proteins get into the central nervous system may help researchers develop ways to block infection with prion proteins.

An abnormal isoform of prion protein is believed to be the infectious agent in transmissible spongiform encephalopathies, and consumption of beef from cattle with bovine spongiform encephalopathy has been linked to new variant Creutzfeldt-Jakob disease in man. But how does rogue get from the gut to the brain to cause disease?

Adriano Aguzzi (University of Zurich, Switzerland) and colleagues report that, at least in mice, lymphohaemopoietic stem cells are the main site of prion replication after intraperitoneal or intravenous inoculation of scrapie prions (Nature 1997; 387: 69‚73). However, these sites are not enough to produce a productive infection of the central nervous system and it seems likely that this requires a chain of tissues expressing endogenous prion proteins. The researchers speculate that the peripheral nervous system could be involved in this chain.

Scientists uncover new CJD clues

September 3, 1997
Patricia Reaney
LONDON -- Adriano Aguzzi and other members of the Institute of Neuropathology at the University of Zurich report in Nature today that prions involved in CJD can travel from the stomach to the spleen and lymphatic system where it accumulates, and that normal prion protein is needed for the abnormal form to replicate and invade the central nervous system and the brain.The story says the Swiss team used mice infected with scrapie to follow the route of prion invasion. Aguzzi was quoted as saying,
"The whole thing is about how the abnormal prion (protein) spreads from one place to another in the body. We are interested in understanding what drives the infective agent, how it gets from the gut all the way up to the brain, the only place where it can do any harm. ... For me, the significance of this work is that this gives us a handle for stopping prion neuro invasion. It gives us a possibility to stop the expression of the normal prion in these specific places and this should effectively prevent the infective agent from reaching the brain. It's early days. We still don't know if we will find a drug to do the job but at least now we have a very distinct idea about what we are searching for..."
Athough CJD is an uncommon disease, with an incubation period ranging from 11 to 71 months, its link to BSE sparked a consumer panic in Britain last year that led to a widespread ban on British beef. The disease is usually not discovered until it is an advanced stage, but Aguzzi said his findings could help people who are newly infected.
"There is a window (of opportunity) during which the (abnormal) prions are in the body but have not yet reached the central nervous system and there I think something can be done," he said.
Neurografts are an interesting technique for exploring the interactions of prions with the brain and lymphatic system, more relevent to real life processes of the epidemic than most of the research we are seeing. seems like expression of prion gene outside the CNS important, suggests that normal role cannot be strictly neuro this or that. i am enclosing what nature had online as well as abstracts of a couple earlier articles on neurografting.

Prp-expressing tissue required for transfer of scrapie infectivity from spleen to brain

Nature 389, 69 (1997)
T Bl”ttler, S Brandner, A J Raeber, M A Klein, T Voigtl”nder,  C Weissmann & A Aguzzi

Much available evidence points to a pathological isoform of the prion protein PrP being the infectious agent that causes transmissible spongiform encephalopathies, but the mechanisms controlling the neurotropism of prions are still unclear. The authors have previously shown that mice that do not express PrP (Prnp o/o mice) are resistant to infection by prions, and that if a Prnp+/+ neurograft is introduced into such animals and these are infected intracerebrally with scrapie, the graft but not the surrounding tissue shows scrapie pathology. Here the authors show that PrP-expressing neurografts in Prnp o/o mice do not develop scrapie histopathology after intraperitoneal or intravenous inoculation with scrapie prions. Prion titres were undetectable in spleens of inoculated Prnp o/o mice, but were restored to wild-type levels upon reconstitution of the host lymphohaemopoietic system with PrP-expressing cells.

Surprisingly, however, i.p. or i.v. inoculation failed to produce scrapie pathology in the neurografts of 27 out of 28 reconstituted animals, in contrast to intracerebral inoculation. They conclude that transfer of infectivity from the spleen to the central nervous system is crucially dependent on the expression of PrP in a tissue compartment that cannot be reconstituted by bone marrow transfer. Thus the requirement for the normal isoform of PrP in peripheral tissues represents a bottleneck for the spread of prions from peripheral sites to the central nervous system.

Normal host prion protein (PrPC) is required for scrapie spread within the CNS

Proc Natl Acad Sci U S A 1996 Nov 12;93(23):13148-13151
Brandner S, Raeber A, Sailer A, Blattler T, Fischer M, Weissmann C, Aguzzi A

Mice devoid of PrPC (Prnp%) are resistant to scrapie and do not allow propagation of the infectious agent (prion). PrPC-expressing neuroectodermal tissue grafted into Prnp% brains but not the surrounding tissue consistently exhibits scrapie-specific pathology and allows prion replication after inoculation. Scrapie prions administered intraocularly into wild-type mice spread efficiently to the central nervous system within 16 weeks. To determine whether PrPC is required for scrapie spread, we inoculated prions intraocularly into Prnp% mice containing a PrP-overexpressing neurograft. Neither encephalopathy nor protease-resistant PrP (PrPSc) were detected in the grafts for up to 66 weeks. Because grafted PrP-expressing cells elicited an immune response that might have interfered with prion spread, we generated Prnp% mice immunotolerant to PrP and engrafted them with PrP-producing neuroectodermal tissue. Again, intraocular inoculation did not lead to disease in the PrP-producing graft. These results demonstrate that PrP is necessary for prion spread along neural pathways.

Tracking prions: the neurografting approach.

Cell Mol Life Sci 1997 Jun;53(6):485-495 Aguzzi A, Blattler T, Klein MA, Raber AJ, Hegyi I, Frigg R, Brandner S, Weissmann C

The physical nature of the agent that causes transmissible spongiform encephalopathies (the 'prion'), is the subject of passionate controversy. Investigation of it has benefited tremendously from the use of transgenic and knockout technologies. However, prion diseases present several other enigmas, including the mechanism of brain damage and how the affinity of the agent for the central nervous system is controlled. Here we show that such questions can be effectively addressed in transgenic and knockout systems, and that pathogenesis may be clarified even before we can be certain about the nature of the infectious agent. Availability of mice overexpressing the Prnp gene (which encodes the normal prion protein) and Prnp knockout mice allows for selective reconstitution experiments aimed at expressing PrP in specific portions of the brain or in selected populations of hemato- and lymphopoietic origin. We summarize how such studies can offer insights into how prions administered to peripheral sites can gain access to central nervous tissue, and into the molecular requirements for spongiform brain damage.

Neuro-immune connection in spread of prions in the body?

Lancet 1997 Mar 15;349(9054):742-743

Aguzzi A

Human rabies -- Montana and Washington, 1997.

MMWR Morb Mortal Wkly Rep 1997 Aug 22;46(33):770-774 
On January 5 and January 18, 1997, respectively, a man in Montana and a man in Washington died of neurologic illnesses initially suspected to be Creutzfeldt-Jakob disease (CJD) but diagnosed as rabies encephalitis during subsequent histologic examination on autopsy. The cases were not linked epidemiologically, and no secondary cases occurred.

Postexposure prophylaxis (PEP) was administered to 113 potential contacts. This report summarizes the clinical presentations of the cases and the epidemiologic investigations by the Montana Department of Public Health and Human Services and the Washington State Department of Health; nucleic acid sequencing indicated that the silver-haired bat (Lasionycteris noctivagans) and the big brown bat (Eptesicus fuscus), respectively, were the probable sources of exposure.

New Kind of Cancer Mutation Found

Trisha Gura  Science 5 Sept 97
[Prion gene is also highly susceptible to DNA slippage bubbles in its repeat region. The observations here also suggest another mechanism for sporadic CJD through somatic mutation. -- webmaster]

...Bert Vogelstein and Kenneth Kinzler at Johns Hopkins University School of Medicine and their colleagues have tracked down a genetic culprit that might explain at least part of the discrepancy--and it works in a way never seen before for any cancer-causing mutation. Paving the way. A single base change--adenine (A) replaces thymine (T)--leads to further mutations that inactivate the APC gene and increase the risk of colon cancer.

In the September issue of Nature Genetics, the researchers report that they have found an inherited mutation in a gene called APC, which normally holds cell growth in check and can cause colon cancers when mutated. But unlike previously identified mutations, the new one does not directly affect the function of the gene. Rather, the mutation may render the surrounding DNA susceptible to mistakes by the enzyme that copies genes when cells replicate, thereby creating new mutations that do lead to loss of gene function.

"This could be a landmark study of a novel mechanism," says molecular biologist Jeffrey Trent of the National Human Genome Research Institute (NHGRI) in Bethesda, Maryland....
A change in the APC gene at first glance appeared to be innocuous--a simple switch from a thymine (T) to an adenine (A) at position 1307 that didn't look like it would disrupt the gene's ability to function. Such gene changes, called polymorphisms, are common.

What raised the researchers' suspicions was a strange phenomenon that occurred when they tested the patient's APC gene in a routine assay that allows it to make its protein product. They found that the protein began to pick up extra mutations in and around the region that contains the T-to-A switch. That apparently happened, Kinzler says, because the mutation creates a stretch of eight consecutive adenines, which are often misread by polymerase enzymes that transcribe genes into messenger RNAs (mRNAs)--the first step toward making proteins.

"The DNA strands can get a little one-to-two base-pair bubble," says Kinzler. "That allows the polymerase to put in an extra base without realizing there is a mistake."
Such "frameshift" mutations can totally garble the rest of the message, creating shortened forms of the protein or rendering it useless. If just mRNA synthesis were affected, the situation might not be harmful, because it wouldn't lead to permanent loss of all functional APC protein. But the same kind of error can also occur in the DNA itself during replication. The Johns Hopkins team found that this may in fact be happening in the colon cells of some patients who have an increased risk of colon cancer but don't carry the original APC mutations. When the investigators looked at the APC gene in tumor and blood samples of these patients, they found that all the tumors that carried the second type of inactivating mutation also had the T-to-A change. Blood cells from the same patients only had the T-to-A switch, however. These results suggest that the patients inherited the base change and developed the other mutations later, but only in the colon cells that became cancerous.

Briefly Noted

Nature Genetics
Alpha-synuclein -- a link between Parkinson and Alzheimer diseases?
Nathanial Heintz & Huda Zoghbi 325

Alpha-Synuclein in Lewy bodies 
8.27 Nature
M G Spillantini, ... & M Goedert 

Efficient assembly of functional cytochrome P450 2C2 requires  a spacer sequence between the N-terminal signal anchor and  catalytic domains
1997 272: J. Biol. Chem. 1997 Sept 5 272(36). 22891. 
Ci-Di Chen, Balraj Doray, Byron Kemper 

Amyloid {beta}-protein fibrillogenesis. Detection of a  protofibrillar intermediate.
1997 272: J. Biol. Chem. 1997 Aug 29 272(35). 22364. 
Dominic M. Walsh, Aleksey Lomakin, George B. Benedek, Margaret M. Condron, David

Misdiagnosis of multiple sclerosis 
August 1, 1997 Lancet
A C Young

Structural biology and phylogenetic estimation 
Nature 25 August 1997
G J P Naylor & W M Brown   527 

N-terminal domains of human copper-transporting adenosine
triphosphatases (the Wilson's and Menkes disease proteins) bind
copper selectively in vivo and in vitro with stoichiometry of one
copper per metal-binding repeat 
1997 272: J. Biol. Chem. 1997 July 25 272(30). 18939. 
Svetlana Lutsenko, ..Conrad T. Gilliam, ...

Crystal Structure of the C-terminal Tetrad Repeat from Synexin 
(Annexin VII)of Dictyostelium discoideum
Journal of Molecular Biology Volume 270 Number 1 p79-88
Susanne Liemann,...,Uwe Jacob

PROTEIN FOLDING: The difference with prokaryotes 
Nature 388, 329-331 (1997)
Mary-Jane Gething 
In prokaryotes, the polypeptide chains in which proteins are synthesized only tend to fold into
their final, operational form when the chain is complete. In eukaryotes, by contrast,
individual domains of a single protein can fold sequentially and independently. This striking
finding can help to resolve the puzzle of why prokaryotes tend to rely much more heavily on
chaperonin- assisted protein folding than do eukaryotes, but the molecular mechanisms
underlying the different treatment of nascent polypeptide chains remain to be defined. 
The evolution of complex genomes requires that new combinations of pre-existing protein
domains successfully fold into modular polypeptides. During eukaryotic translation model
two-domain polypeptides fold efficiently by sequential and co-translational folding of their
domains. In contrast, folding of the same proteins in Escherichia coli is post- translational,
and leads to intramolecular misfolding of concurrently folding domains. Sequential domain
folding in eukaryotes may have been critical in the evolution of modular polypeptides, by
increasing the probability that random gene-fusion events resulted in immediately foldable
protein structures. 
Recombination of protein domains facilitated by co-translational folding in eukaryotes
Nature 388, 343 (1997)
 W J Netzer & F U Hartl 

Prion proteins - evolution and preservation of secondary structure

FEBS Letters 1997; 412(N3): 429-32 JA 1997
Kuznetsov,I.B.; Morozov,P.S.; Matushkin,Y.G.
Prions cause a variety of neurodegenerative disorders that seem to result from a conformational change in the prion protein (PrP). Thirty-two PrPsequences have been subjected to phylogenetic analysis followed by reconstructionof the most probable evolutionary spectrum of amino acid replacements. Their placement rates suggest that the protein does not seem to be very conservative, but in the course of evolution amino acids have only been substituted within the elements of the secondary structure by those with very similar physico-chemical properties. Analysis of the full spectrum of single-step amino acid substitutions in human PrP using secondary structure prediction algorithms shows an over-representation of substitutions that tend to destabilize alpha-helices.

Incompatibility gene of the fungus Podospora anserina behaves as a prion analog

Proc. Natl. Acad. Sci. USA free full text
Vol. 94, pp. 9773-9778, September 1997
Virginie Coustou, Carol Deleu, Sven Saupe, and Joel Begueret
The het-s locus of Podospora anserina is a heterokaryon incompatibility locus. The coexpression of the antagonistic het-s and het-S alleles triggers a lethal reaction that prevents the formation of viable heterokaryons. Strains that contain the het-s allele can display two different phenotypes, [Het-s] or [Het-s*], according to their reactivity in incompatibility. The detection in these phenotypically distinct strains of a protein expressed >from the het-s gene indicates that the difference in reactivity depends on a posttranslational difference between two forms of the polypeptide encoded by the het-s gene.

This posttranslational modification does not affect the electrophoretic mobility of the protein in SDS/PAGE. Several results suggest a similarity of behavior between the protein encoded by the het-s gene and prions. The [Het-s] character can propagate in [Het-s*] strains as an infectious agent, producing a [Het-s*] [Het-s] transition, independently of protein synthesis. Expression of the [Het-s] character requires a functional het-s gene. The protein present in [Het-s] strains is more resistant to proteinase K than that present in [Het-s*] mycelium. Furthermore, overexpression of the het-s gene increases the frequency of the transition from [Het-s*] to [Het-s]. We propose that this transition is the consequence of a self-propagating conformational modification of the protein mediated by the formation of complexes between the two different forms of the polypeptide.


The het-s locus of the filamentous fungus Podospora anserina is one of the nine known loci controlling heterokaryon incompatibility in that species (for review, see ref. 1). Coexpression of the antagonistic het-s and het-S alleles in the same cytoplasm triggers an adverse reaction that prevents the formation of viable heterokaryotic cells between strains that contain the incompatible alleles (2). This locus encodes a 289-aa protein that is not essential for cell viability or completion of the life cycle of the fungus (3, 4). The proteins encoded by the incompatible alleles differ by 14 amino acid substitutions, but one difference is sufficient for expression of the antagonistic [Het-s] and [Het-S] specificities (5).

It has been reported by Rizet (ref. 2; for review, see ref. 6) that haploid strains of the het-s genotype can exhibit two different phenotypes: they either are incompatible with het-S strains (this phenotype will herein be designated [Het-s]), or they are neutral in incompatibility and display the [Het-s*] phenotype. This latter phenotype is stably maintained during vegetative growth but a transition from [Het-s*] to [Het-s] can occur spontaneously at a very low frequency, estimated under 107 per nucleus (7). This phenotypic conversion is, however, invariably induced after anastomosis and cytoplasmic mixing with a [Het-s] strain. This transition cannot be induced by strains that contain het-S or the null alleles het-sx and het-sƒ. The [Het-s] character is dominant and can propagate in [Het-s*] strains in the absence of nuclear transmission. When it has been induced, the [Het-s*] [Het-s] transition spreads very rapidly as an infectious process >from the region of anastomosis throughout the mycelium. Both the [Het-s] and [Het-s*] characters are transmitted as nonmendelian elements through meiosis. The phenotype of the offsprings of the het-s genotype depends on the phenotype of the female parent, suggesting that these phenotypes are controlled by cytoplasmic elements. It has been proposed >from these different results that the expression of the het-s gene might be positively controlled by its protein product (7).

NMR characterization of the full-length recombinant murine prion protein, mPrP.

FEBS Lett 1997 Aug 18;413(2):282-288
Riek R, Hornemann S, Wider G, Glockshuber R, Wuthrich K
...NMR experiments showed that the previously determined globular three-dimensional structure of the C-terminal domain mPrP(121-231) is preserved in the intact protein, and that the N-terminal polypeptide segment 23-120 is flexibly disordered. This structural information is based on nearly complete sequence-specific assignments for the backbone amide nitrogens, amide protons and alpha-protols of the polypeptide segment of residues 121-231 in mPrP(23-231)....

The linewidths in heteronuclear 1H-15N correlation spectra and 15N[1H]-NOEs showed that the well structured residues 126-230 have correlation times of several nanoseconds, as is typical for small globular proteins, whereas correlation times shorter than 1 nanosecond were observed for all residues of mPrP(23-231) outside of this domain.

Recombinant full-length murine prion protein, mPrP(23-231): purification and spectroscopic characterization.

FEBS Lett 1997 Aug 18;413(2):277-281 
Hornemann S, Korth C, Oesch B, Riek R, Wider G, Wuthrich K, Glockshuber R
The cellular prion protein of the mouse, mPrP(C), consists of 208 amino acids (residues 23-231). It contains a carboxy-terminal domain, mPrP(121-231), which represents an autonomous folding unit with three alpha-helices and a two-stranded antiparallel beta-sheet. We expressed the complete amino acid sequence of the prion protein, mPrP(23-231), in the cytoplasm of Escherichia coli. mPrP(23-231) was solubilized from inclusion bodies by 8 M urea, oxidatively refolded and purified to homogeneity by conventional chromatographic techniques.

Comparison of near-UV circular dichroism, fluorescence and one-dimensional 1H-NMR spectra of mPrP(23-231) and mPrP(121-231) shows that the amino-terminal segment 23-120, which includes the five characteristic octapeptide repeats, does not contribute measurably to the manifestation of three-dimensional structure as detected by these techniques, indicating that the residues 121-231 might be the only polypeptide segment of PrP(C) with a defined three-dimensional structure.

Prion proteins: evolution and preservation of secondary structure.

FEBS Lett 1997 Aug 4;412(3):429-432
Kuznetsov IB, Morozov PS, Matushkin YG
Prions cause a variety of neurodegenerative disorders that seem to result from a conformational change in the prion protein (PrP). Thirty-two PrP sequences have been subjected to phylogenetic analysis followed by reconstruction of the most probable evolutionary spectrum of amino acid replacements. The replacement rates suggest that the protein does not seem to be very conservative, but in the course of evolution amino acids have only been substituted within the elements of the secondary structure by those with very similar physico-chemical properties. Analysis of the full spectrum of single-step amino acid substitutions in human PrP using secondary structure prediction algorithms shows an over-representation of substitutions that tend to destabilize alpha-helices.

Neurofibrillary tangles in GSS with the A117V prion gene mutation

J Neurol Neurosurg Psychiatry 1997 Aug;63(2):240-246
Tranchant C, Sergeant N, Wattez A, Mohr M, Warter JM, Delacourte A
One patient of a French family with Gerstmann-Straussler-Scheinker syndrome with the mutation in codon 117 of the prion protein (PrP) gene displayed unexpected neuritic degeneration around PrP plaques and numerous diffuse neurofibrillary tangles, whereas other members did not. The tau profile in this patient's brain was analysed and compared with one from another member of the Gerstmann-Straussler-Scheinker family as well as with the Alzheimer's tau profile. A panel of well characterised antibodies against both normal tau protein and paired helical filaments-tau protein was used on immunoblots of brain proteins resolved by mono and two dimensional gels.

The tau profile in the patient with Gerstmann-Straussler-Scheinker syndrome without neurofibrillary tangles was normal. The tau profile from the patient with GSS and neurofibrillary tangles was characterised by a hyperaggregation state of tau protein. This case illustrates the phenotypic heterogeneity of the GSS117 mutation not only from one family to another, but also between members of the same family. In this family, the presence of neurofibrillary tangles is still unexplained, but could be correlated with either the protracted duration of the disease or the old age of the patient.

A neurotoxic and gliotrophic fragment of the prion protein increases plasma-membrane microviscosity

Neurobiology of Disease 1997; 4(N1): 47-57 1997
Salmona,M.; Forloni,G.; Diomede,L.; Algeri,M.; .. Tagliavini,F.; Bugiani,O.
Prion-related encephalopathies are characterized by astrogliosis and nerve cell degeneration and loss. These lesions might be the consequence of an interaction between the abnormal isoform of the cellular prion protein that accumulates in nervous tissue and the plasma membranes. Previously we found that a synthetic peptide, homologous to residues 106-126 of the human prion protein, is fibrillogenic and toxic to neurons and trophic to astrocytes in vitro. This study dealt with the ability of the peptide to interact with membranes.

Accordingly, we compared PrP 106-126 with different synthetic PrP peptides (PrP 89-106, PrP 127-147, a peptide with a scrambled sequence of 106-126, and PrP 106-126 amidated at the C-terminus) as to the ability to increase the microviscosity of artificial and natural membranes. The first three had no effect on nerve and glial cells in vitro, whereas the amidated peptide caused neuronal death. Using a fluorescent probe that becomes incorporated into the hydrocarbon core of the lipid bilayer and records the lipid fluidity, we found PrP 106-126 able to increase significantly the membrane microviscosity of liposomes and of all cell lines investigated. This phenomenon was associated with the distribution of the peptide over the cell surface, but not with changes in the membrane lipid or protein content, or with membrane lipid phase transitions.

Accordingly we deduced that increased membrane microviscosity was unrelated to changes in the membrane native components and was the result of increased lipid density following PrP 106-126 embedding into the lipid bilayer. No control peptides had comparable effects on the membrane microviscosity, except PrP 106-126 amidated at the C-terminus. Since the latter was as neurotoxic, but not as fibrillogenic, as PrP 106-126, we argued that the ability of PrP 106-126 to increase membrane microviscosity was unrelated to the propensity of the peptide to raise fibrils. Rather, it could be connected with the primary structure of PrP 106-126, characterized by two opposing regions, one hydrophilic and the other hydrophobic, that enabled the peptide to interact with the lipid bilayer.

Based on these findings, we speculated that the glial and nerve cell involvement occurring in prion-related encephalopathies might be caused by the interaction with the plasma membrane of a PrP 106-126-like fragment or of the sequence spanning residues 106-126 of the abnormal isoform of the prion protein.

Acceleration of Amyloid Fibril Formation by Specific Binding of A-(1-40) Peptide to Ganglioside-containing Membrane Vesicles

JBC Volume 272, Number 37, Issue of September 12, 1997 pp. 22987-22990
Lin-P'ing Choo-Smith  , William Garzon-Rodriguez ß , Charles G. Glabe ß and Witold K. Surewicz
The interaction of Alzheimer's A peptide and its fluorescent analogue with membrane vesicles was studied by spectrofluorometry, Congo Red binding, and electron microscopy. The peptide binds selectively to the membranes containing gangliosides with a binding affinity ranging from 106 to 107 M depending on the type of ganglioside sugar moiety. This interaction appears to be ganglioside-specific as under our experimental conditions (neutral pH, physiologically relevant ionic strength), no A binding was observed to ganglioside-free membranes containing zwitterionic or acidic phospholipids. Importantly, the addition of ganglioside-containing vesicles to the peptide solution dramatically accelerates the rate of fibril formation as compared with that of the peptide alone. The present results strongly suggest that the membrane-bound form of the peptide may act as a specific "template" (seed) that catalyzes the fibrillogenesis process in vivo.

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