Identification of candidate proteins binding to prion protein.
CJD stats from Germany
Parvalbumin-immunoreactive neurons associated with CJD
Increased calcium in microglia activated by prion
Astrocyte expression of prion protein
Prion research: the next frontiers
Cerebellar leucine-rich ANP interacts with ataxin-1 of SCA-1
Angiopathies masquerading as Alzheimer's disease?
Blood brain barrier in Alzheimer
Alzheimer's disease: The ins and outs of amyloid-beta
New gene linked to Alzheimer's is found
C. elegans presenilin functionally redundant with presenilin
De novo appearance of PSI+ prion in S. cerevisiae
Control of scrapie eventually possible?
Further transmission of CJD via corneal transplant.
Differential resistance to proteinase K of yeast Ure2p in vitro
Dtsch Med Wochenschr 1997 Sep 12;122(37):1099-1105 [Article in German] Poser S, Zerr I, Schulz-Schaeffer WJ, Kretzschmar HA, Felgenhauer KProspective epidemiological studies are being employed to determine the incidence and possible risk factors of CJD in five European countries in which bovine spongiform encephalopathy (BSE) occurs at different rates of incidence.
Using a voluntary reporting system throughout the Federal Republic of Germany, suspected cases of CJD were investigated and the incidence calculated. Possible risk factors in patients and control groups were obtained by questionnaire. Serum and cerebrospinal fluid samples served to delineate genetic forms and distinguish the disease from other major dementias.
A total of 544 patients with suspected CJD, reported in Germany between 1993 and 1997, were examined. 232 (plus 27 investigated only neuropathologically) were confirmed as definite or probable, an annual incidence per million population of between 0.76 (for 1994) and 0.98 (for 1995), similar to figures from other European countries. In Great Britain, the cases of "new variant" CJD, not yet observed in Germany, were excluded from the calculation of incidence. So far, dementia in the family and handling of horn shavings have been identified as risk factors.
A rise in the concentrations of neurone-specific enolase and of S100 protein as well as the demonstration of certain proteins in cerebrospinal fluid (p130/ 131 and 14-3-3, respectively) have been shown as being diagnostically superior to EEG changes.
Nature 389, 966 (1997) P J Skinner, B T Koshy, C J Cummings, I A Klement, K Helin, A Servadio, H Y Zoghbi & H T OrrAtaxin-1 with an expanded glutamine tract alters nuclear matrix-associated structures Spinocerebellar ataxia type 1 (SCA1) is one of several neurodegenerative disorders caused by an expansion of a polyglutamine tract. It is characterized by ataxia, progressive motor deterioration, and loss of cerebellar Purkinje cells. To understand the pathogenesis of SCA1, the authors examined the subcellular localization of wild-type human ataxin-1 (the protein encoded by the SCA1 gene) and mutant ataxin-1 in the Purkinje cells of transgenic mice.
They found that ataxin-1 localizes to the nuclei of cerebellar Purkinje cells. Normal ataxin-1 localizes to several nuclear structures ~0.5 Ám across, whereas the expanded ataxin-1 localizes to a single ~2-Ám structure, before the onset of ataxia. Mutant ataxin-1 localizes to a single nuclear structure in affected neurons of SCA1 patients. Similarly, COS-1 cells transfected with wild-type or mutant ataxin-1 show a similar pattern of nuclear localization; with expanded ataxin-1 occurring in larger structures that are fewer in number than those of normal ataxin-1.
Colocalization studies show that mutant ataxin-1 causes a specific redistribution of the nuclear matrix-associated domain containing promyelocytic leukaemia protein. Nuclear matrix preparations demonstrate that ataxin-1 associates with the nuclear matrix in Purkinje and COS cells. They therefore propose that a critical aspect of SCA1 pathogenesis involves the disruption of a nuclear matrix-associated domain.
Nature 389, 971 (1997) A Matilla, B T Koshy, C J Cummings, T Isobe, H T Orr & H Y ZoghbiThe cerebellar leucine-rich acidic nuclear protein interacts with ataxin-1 Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder characterized by ataxia, progressive motor deterioration, and loss of cerebellar Purkinje cells. SCA1 belongs to a growing group of neurodegenerative disorders caused by expansion of CAG repeats, which encode glutamine. Although the proteins containing these repeats are widely expressed, the neurodegeneration in SCA1 and other polyglutamine diseases selectively involves a few neuronal subtypes. The mechanism(s) underlying this neuronal specificity is unknown.
Here the authors show that the cerebellar leucine-rich acidic nuclear protein (LANP) interacts with ataxin-1, the SCA1 gene product. LANP is expressed predominantly in Purkinje cells, the primary site of pathology in SCA1. The interaction between LANP and ataxin-1 is significantly stronger when the number of glutamines is increased. Immunofluorescence studies demonstrate that both LANP and ataxin-1 colocalize in nuclear matrix-associated subnuclear structures. The features of the interaction between ataxin-1 and LANP, their spatial and temporal patterns of expression, and the colocalization studies indicate that cerebellar LANP is involved in the pathogenesis of SCA1.
Proc. Natl. Acad. Sci. USA, Vol. 94, pp. 12204-12209, October 1997 Xiajun Li and Iva GreenwaldMutant presenilins have been found to cause Alzheimer disease. Here, we describe the identification and characterization of HOP-1, a Caenorhabditis elegans presenilin that displays much more lower sequence identity with human presenilins than does the other C. elegans presenilin, SEL-12. Despite considerable divergence, HOP-1 appears to be a bona fide presenilin, because HOP-1 can rescue the egg-laying defect caused by mutations in sel-12 when hop-1 is expressed under the control of sel-12 regulatory sequences. HOP-1 also has the essential topological characteristics of the other presenilins.
Reducing hop-1 activity in a sel-12 mutant background causes synthetic lethality and terminal phenotypes associated with reducing the function of the C. elegans lin-12 and glp-1 genes. These observations suggest that hop-1 is functionally redundant with sel-12 and underscore the intimate connection between presenilin activity and LIN-12/Notch activity inferred from genetic studies in C. elegans and mammals.
Genetic linkage studies have identified a number of loci associated with familial Alzheimer disease (1). Two of these loci encode related multipass transmembrane proteins, presenilins 1 and 2 (PS1 and PS2). Mutations in the genes encoding PS1 and PS2 loci are dominant and fully penetrant for early onset Alzheimer disease (2-4). The presenilins are ubiquitously expressed (3, 4) and found in conjunction with intracellular membranes (5). However, the normal role of presenilins, and the mechanism by which mutant presenilins cause Alzheimer disease, are not known.
Genetic studies in simple organisms offer a powerful approach to understanding the normal role of presenilins. The Caenorhabditis elegans sel-12 gene encodes a protein that displays about 50% amino acid sequence identity to PS1 and PS2 (6). Genetic analysis established that reducing or eliminating sel-12 activity causes an egg-laying defective (Egl) phenotype, and that sel-12 activity facilitates the activity of LIN-12 and GLP-1, two receptors of the LIN-12/Notch family (6). SEL-12 appears to be a bona fide presenilin, because human PS1 and PS2 can rescue the Egl phenotype of a sel-12 mutant (7). Furthermore, the membrane topology of SEL-12 and PS1 appears to be similar (8-10). In addition to the functional and structural similarities, expression studies indicate that SEL-12 and human presenilins are expressed throughout development in many different cell types (3, 4, 7).
We have identified another candidate C. elegans presenilin based on predicted amino acid sequence by searching the genomic sequence database (11, 12). Here, we show that this gene, which we have named hop-1 (hop = homolog of presenilin), encodes a functional presenilin, by demonstrating that HOP-1 can rescue the egg-laying defect of a sel-12 mutant. We also show that HOP-1 has characteristic features of presenilin membrane topology. Finally, we show that reducing hop-1 activity in a sel-12 mutant background results in novel phenotypes, suggesting that hop-1 and sel-12 are functionally redundant.
Vet Q 1997 Sep;19(3):105-113 Schreuder BE, van Keulen LJ, Smits MA, Langeveld JP, Stegeman JAAfter a brief description of the scrapie situation in the Netherlands, the technical progress made in aspects of scrapie diagnosis is reported. Emphasis is placed on the use of immuno-histochemistry (IHC) in the post-mortem histological diagnosis and on the recently published preclinical test for scrapie, in which IHC is applied to tonsillar biopsies. These two approaches use the same IHC technique and enable us to confirm suspected scrapie in individual animals, and for certain genotypes even in the preclinical phase. The tonsillar biopsy method could eventually lead to an infection- or PrPSc-detection method at flock level. Further work is required, including validation of the assay for various breeds, genotypes, and strains of the agent, and the conversion of the test into a more practical assay. The article continues with a discussion of several scrapie control strategies, describing briefly schemes tried in various countries, and elaborates on a proposed scrapie control scheme that could be suitable for the Netherlands. This scheme is essentially based on breeding for resistance, based on PrP genotyping.
J Neurol Neurosurg Psychiatry 1997 Sep;63(3):388-390 Heckmann JG, Lang CJ, Petruch F, Druschky A, Erb C, Brown P, Neundorfer BA 45 year old woman is reported who initially presented with a cerebellar syndrome, severe ataxia, and dysarthria. She rapidly deteriorated to coma vigile with bilateral myoclonic jerks, flexion rigidity, and immobility necessitating complete nursing. Her EEG showed generalised slow activity and periodic biphasic and triphasic waves. The CSF concentration of neuron specific enolase was very high. Consequently the diagnosis of Creutzfeldt-Jakob disease was established. Eight months later she died of respiratory complications. Thirty years earlier the patient had undergone corneal transplantation for keratoconus. Review of the organ donor's hospital records showed that death was caused by intercurrent pneumonia subsequent to subacute spongiform encephalopathy confirmed by necropsy. In view of two previous case reports in the literature it is presumed that the cadaveric cornea was the source of transmission of Creutzfeldt-Jakob disease in this patient.
FEBS Lett 1997 Sep 22;415(1):6-10 Komar AA, Lesnik T, Cullin C, Guillemet E, Ehrlich R, Reiss CThe Ure2p yeast prion-like protein was translated in vitro in the presence of labeled [35S]methionine in either rabbit reticulocyte lysate (RRL) or wheat germ extract (WGE) cell-free systems. When subjected to proteinase K digestion, the Ure2p protein synthesized in WGE was proteolysed much more slowly compared to that synthesized in RRL; this displays fragments of about 31-34 kDa, persisting over 8 min. Thus, the digestion rate and pattern of the protein synthesized in WGE, unlike that synthesized in RRL, revealed characteristic features of the [URE3] prion-like isoform of the Ure2p protein [Masison, D.C. and Wickner, R.B. (1995) Science 270, 93-95].
Chloramphenicol acetyltransferase, synthesized under the same conditions, differed fundamentally in its proteolytic sensitivity toward proteinase K (PK); in the RRL system it was more slowly digested than in WGE, proving specific PK inhibitors to be absent in both systems. Posttranslational addition of the WGE to the RRL-synthesized Ure2p does not protect Ure2p from efficient PK degradation either. The differences in Ure2p degradation may be ascribed to a specific structure or specific states of association of Ure2p synthesized in WGE; obviously, they yield a protein that mimics the behavior of the Ure2p in [URE3] yeast strains. The present data suggest that particular conditions of the Ure2p protein translation and/or certain cellular components (accessory proteins and extrinsic factors), as well as the nature of the translation process itself, could affect the intracellular folding pathway of Ure2p leading to the de novo formation of the prion [URE3] isoform.
[Medline] Cohen DL, Hedera P, Premkumar DR, Friedland RP, Kalaria RNCurrent evidence from genetic and epidemiological studies supports the view that Alzheimer's disease (AD) is a heterogeneous disorder. While the disease is pathologically defined by the presence of specified lesions in form of amyloid plaques and neurofibrillary tangles within the parenchyma, other features of pathology are often either neglected or considered coincidental. Our studies suggest that cerebrovascular pathology is inherently part of the disorder, which could be an important factor in a cause or effect manner.
We have recently identified subjects having died with severe amyloid beta (A beta) protein cerebral amyloid angiopathy (CAA) in the absence of a profound Alzheimer pathology. These subjects, diagnosed with dementia had a late onset disease and were found at autopsy to exhibit severe CAA but paucity of typical AD changes.
Immunocytochemical studies showed numerous microvascular abnormalities as well as characteristic degeneration of the vascular smooth muscle in both surface and intracortical vessels. The pathology was also characterized by occasional intracerebral hemorrhages and multiple infarcts. Further assessment of the abnormalities and amyloid infiltrated cerebral vessels with antibodies to the carboxyl terminus of A beta indicated that the longer, more pathogenic form of A beta(1-42) was found to be highly associated with intracerebral hemorrhages. Our observations suggest that these mild AD cases with a predominantly vascular pathology are variants of AD and bear resemblance to the familial Dutch and Flemish versions of cerebral amyloidosis. We propose that AD is a group of diseases with a variable pathology analogous to the prion diseases, in which a vascular variant also exists.
Genetics 1997 Oct;147(2):507-519 Derkatch IL, Bradley ME, Zhou P, Chernoff YO, Liebman SWIt has previously been shown that yeast prion [PSI+] is cured by GuHCl, although reports on reversibility of curing were contradictory. Here we show that GuHCl treatment of both [PSI+] and [psi-] yeast strains results in two classes of [psi-] derivatives: Pin+, in which [PSI+] can be reinduced by Sup35p overproduction, and Pin-, in which overexpression of the complete SUP35 gene does not lead to the [PSI+] appearance. However, in both Pin+ and Pin- derivatives [PSI+] is reinduced by overproduction of a short Sup35p N-terminal fragment, thus, in principle, [PSI+] curing remains reversible in both cases. Neither suppression nor growth inhibition caused by SUP35 overexpression in Pin+ [psi-] derivatives are observed in Pin- [psi-] derivatives.
Genetic analyses show that the Pin+ phenotype is determined by a non-Mendelian factor, which, unlike the [PSI+] prion, is independent of the Sup35p N-terminal domain. A Pin- [psi-] derivative was also generated by transient inactivation of the heat shock protein, Hsp104, while [PSI+] curing by Hsp104 overproduction resulted exclusively in Pin+ [psi-] derivatives. We hypothesize that in addition to the [PSI+] prion-determining domain in the Sup35p N-terminus, there is another self-propagating conformational determinant in the C-proximal part of Sup35p and that this second prion is responsible for the Pin+ phenotype.
J Neuropathol Exp Neurol 1997 Oct;56(10):1119-1124 Guentchev M, Hainfellner JA, Trabattoni GR, Budka HThere is a distinctive pattern of hippocampal involvement in Creutzfeldt-Jakob disease (CJD) and evidence for selective vulnerability of GABAergic neurons in experimental and human prion disease. We studied hippocampus and temporal cortex from human CJD and control autopsy brains and surgical cryptogenic temporal lobe epilepsy specimens for distribution and density of parvalbumin (PV) and calbindin-D28K (Cal) -positive neurons that are sub-populations of GABAergic neurons.
Pathology was evaluated semiquantitatively in 8 regions in 23 CJD brains for severity of spongiform change, astrogliosis and pathological prion protein deposition. In CJD, pathology was severe in pre-parasubiculum and temporal cortex, and little or absent in CA1-4; PV+ neurons were severely reduced or absent in all cases, whereas Cal+ neurons were largely preserved. In controls, the density of PV+ neurons was highest in pre-parasubiculum and temporal cortex, and lowest in CA1-4. In cTLE, loss of PV+ neurons was seen only in CA1-4. The diffuse and severe loss of PV+ neurons in CJD, and the topographical correlation of tissue lesioning in CJD with density of PV+ neurons in controls suggest selective vulnerability and early loss of this subset of inhibitory neurons in CJD. This might relate to characteristic CJD symptoms such as myoclonus and the distinctive EEG pattern.
Nature 389, 795-798 (1997) -- offline Adriano Aguzzi and Charles WeissmannAt the beginning of this month, Stanley Prusiner of the University of California, San Francisco, was awarded the Nobel Prize in Physiology or Medicine for his work on the infectious agent that causes spongiform encephalopathies -- the prion. His 'protein-only' theory now has many advocates, and the advances that have been made, the questions that remain and the ways in which these could be addressed are discussed in this feature.
Nature 389, 677-678 (1997) Konrad Beyreuther and Colin L. MastersThe extracellular plaques of amyloid-beta (A-beta) peptide that form in the brains of pateints with Alzheimer's disease are thought to be involved in neurodegeneration but, until now, nobody was sure how A-beta interfered with neuronal function. The finding that A-beta interacts with the newly discovered endoplasmic-reticulum-associated binding protein (ERAB) in neurons may help to solve this puzzle.
Amyloid-beta is a neurotoxic peptide which is implicated in the pathogenesis of Alzheimers disease. It binds an intracellular polypeptide known as ERAB, thought to be a hydroxysteroid dehydrogenase enzyme, which is expressed in normal tissues, but is overexpressed in neurons affected in Alzheimers disease. ERAB immunoprecipitates with amyloid-beta, and when cell cultures are exposed to amyloid-beta, ERAB inside the cell is rapidly redistributed to the plasma membrane. The toxic effect of amyloid-beta on these cells is prevented by blocking ERAB and is enhanced by overexpression of ERAB. By interacting with intracellular amyloid-beta, ERAB may therefore contribute to the neuronal dysfunction associated with Alzheimers disease.
Mus musculus amyloid beta-peptide binding protein (ERAB) complete cds. ACCESSION U96116 MCKMAAAVRSVKGLVAVVTGGASGPWLATAKRLVGQGATAVLLD VPDSEGESQAKKLGESCIFAPANVTSEKEIQAALTLAKEKFGRIDVAVNCAGIAVAIK TYHQKKNKIHTLEDFQRVINVNLIGTFNVIRLVAGEMGQNEPDQGGQRGVIINTASVA AFEGQVGQAAYSASKGGIDGMTLPIARDLAPTGIRVVTIAPGLFATPLLTTLPEKVRN FLASQVPFPSRLGDPAEYAHLVQTIIENPFLNGEVIRLDGAIRMQP
Nature 389, 689 (1997) S D Yan, J Fu, ... M Tohyama, S Ogawa, A Roher & D Stern
Ann N Y Acad Sci 1997 Sep 26;826:161-172 Wisniewski HM, Vorbrodt AW, Wegiel JEvidence is accumulating that suggests that increased permeability of the BBB to blood-borne proteins is favorable for the development of neuropathologic changes such as amyloid angiopathy and formation of amyloid plaques in the AD brain. To study this problem, we applied a quantitative immunocytochemical procedure that enables evaluation of the barrier function of brain microvasculature to endogenous albumin. This procedure was successfully used on scrapie-infected mice, which represent a unique animal model enabling study of an interrelation between BBB function and deposition of amyloid within vascular wall and neuritic plaques.
Biopsy specimens obtained during neurosurgical procedures (tumors and dementia) were also examined. Our observations indicate that (1) the vast majority of brain microvessels in scrapie-infected mice and in demented individuals show normal features of the BBB; (2) only those microvascular segments directly surrounded by amyloid plaques or representing amyloid angiopathy show increased permeability to endogenous albumin; (3) numerous immunosignals over the amyloid deposits in plaques and in the wall of angiopathic vessels suggest the affinity of extravasated albumin to the amyloid material.
October 14 1997 BY NIGEL HAWKES Times Science editorOXFORD scientists have discovered a gene that multiplies the risk of getting Alzheimer's disease. People over the age of 65 who carry the new gene and another Alzheimer's gene ? about 6 per cent of the white population ? have a 30 times greater chance of developing the disease than those without it. David Smith, whose team at the Department of Pharmacology made the finding, published today in the journal Human Molecular Genetics, said that the products of the two genes combine to increase the danger.
"If we can understand how the interaction works it might be possible to develop a drug that interferes with it," he said. But he thought it might take 20 years before a treatment became available.
Screening people at high risk would be pointless in the absence of an effective treatment, he said. The type of Alzheimer's involved is that which develops in old age, not the early-onset version, which seems to have different causes.
Associated Press October 14, 1997CHICAGO -- Researchers believe they have found the hiding place of a gene that makes certain people susceptible to Alzheimer's disease after age 60. Evidence that the gene may lurk somewhere within a region of chromosome 12, one of the string-like structures that carry genes, was reported in Wednesday's issue of The Journal of the American Medical Association.
"We've identified a certain section of chromosome 12, and the gene has to be in there somewhere," said the study's co-author, Jonathan Haines, director of the human genetics program at Vanderbilt University. "We've narrowed it down at least one-hundredfold."Genetic links to early-onset of Alzheimer's have been established, but the disease typically affects people over 65. On Monday, researchers at Oxford University reported they have discovered a gene that may play a critical role in the emergence of late-onset Alzheimer's.
The findings on chromosome 12 appear to identify a new genetic risk factor, said Dr. Allen Roses, one of the researchers and the worldwide director of genetics for Glaxo Wellcome pharmaceuticals. Researchers closed in on its location by doing a wide-ranging scan of all the chromosomes from members of 16 families. The evidence of the suspect region was confirmed in a second group of 38 families.
Despite major advances in the last decade, researchers say 50 percent of the genetic markers for Alzheimer's -- the nation's fourth-leading cause of death -- remain a mystery. The disease afflicts 4 million Americans.
Neurobiol-Dis. 1997; 3(4): 339-55 Yehiely-F; Bamborough-P; Da-Costa-M; Perry-BJ; Thinakaran-G; Cohen-FE; Carlson-GA; Prusiner-SBPrion diseases are disorders of protein conformation that produce neurodegeneration in humans and animals. Studies of transgenic (Tg) mice indicate that a factor designated protein X is involved in the conversion of the normal cellular prion protein (PrPC) into the scrapie isoform (PrPSc); protein X appears to interact with PrPC but not with PrPSc. To search for PrPC binding proteins, we fused PrP with alkaline phosphatase (AP) to produce a soluble, secreted probe. PrP-AP was used to screen a lambdagt11 mouse brain cDNA library, and six clones were isolated.
Four cDNAs are novel while two clones are fragments of Nrf2 (NF-E2 related factor 2) transcription factor and Aplp1 (amyloid precursor-like protein 1). The observation that PrP binds to a member of the APP (amyloid precursor protein) gene family is intriguing, in light of possible relevance to Alzheimer's disease. Four of the isolated clones are expressed preferentially in the mouse brain and encode a similar motif.
EMBO J 1997 Oct 15;16(20):6057-6065 Raeber AJ, Race RE, Brandner S, Priola SA, Sailer A, Bessen RA, Mucke L, Manson J, Aguzzi A, Oldstone MBA, Weissmann C, Chesebro BTransmissible spongiform encephalopathies are characterized by spongiosis, astrocytosis and accumulation of PrPSc, an isoform of the normal host protein PrPC. The exact cell types responsible for agent propagation and pathogenesis are still uncertain. To determine the possible role of astrocytes, we generated mice devoid of murine PrP but expressing hamster PrP transgenes driven by the astrocyte-specific GFAP promoter. After inoculation with hamster scrapie, these mice accumulated infectivity and PrPSc to high levels, developed severe disease after 227 ▒ 5 days and died 7 ▒ 4 days later. Therefore, astrocytes could play an important role in scrapie pathogenesis, possibly by an indirect toxic effect on neurons. Interestingly, mice expressing the same transgenes but also endogenous murine PrP genes propagated infectivity without developing disease.
Spongiosis, astrocytosis and accumulation of PrPSc are the most prominent neuropathological features associated with TSEs; neuronal loss is not consistently found in mouse scrapie (Scott and Fraser, 1984; S.Brandner, unpublished observations). While neurons are clearly affected in the disease process, it is still unclear whether other brain cells also play a role. Recently it was demonstrated that neuron-specific expression of HaPrP driven by the NSE promoter in transgenic mice is sufficient to mediate susceptibility to hamster scrapie (Race et al., 1995). However, astrocytes may also participate in scrapie pathogenesis because these cells show marked morphological abnormalities early in the disease process (Pattison, 1965; Eklund et al., 1967), and may in some cases be the earliest site of accumulation of PrPSc in the brain (Diedrich et al., 1991).
In the present paper we report on transgenic mice expressing HaPrP under control of the GFAP promoter. The GFAP expression vector used for generating GFAP-HaPrP transgenic mice was shown previously to confer astrocyte-specific expression upon a lacZ reporter transgene (Mucke et al., 1991). In these experiments, lacZ expression was restricted to astrocytes in the CNS and lens epithelium, and was not observed in neurons or in other tissues including spleen, lymph node and sciatic nerve. In the GFAP-HaPrP mice described in the present report, HaPrP protein was found only in the brain and was below detection levels in other tissues. Although the constitutive level of HaPrP in the brains of transgenic mice was only ~5% of that observed in normal hamster brain, the GFAP-HaPrP transgene was up-regulated after cryolesioning and after infection with hamster scrapie. Within the limits of detection in vivo, HaPrP mRNA co-localized exclusively with GFAP-positive cells in brain sections of Tg3/Prnp0/0 mice, as expected from the astrocyte specificity of the GFAP promoter. There was no evidence for HaPrP expression in neurons, and if any occurred, it would be at very low levels and unlikely to contribute to pathogenesis. No HaPrP protein was seen in spleen by immunoblotting, although HaPrP mRNA was detected at low levels.
Inoculation of Tg3/Prnp0/0 mice with hamster scrapie agent resulted in neurological disease after a relatively long incubation time of 227 days. This was not unexpected in view of the dependence of incubation time on PrPC expression levels. More surprising was the rapid progression of the clinical phase leading to death of all animals within about a week. This might be explained by the inducible nature of the GFAP-HaPrP transgene leading to increased levels of HaPrPC after astrocyte activation, thereby providing more substrate for the conversion of HaPrPC into HaPrPSc.
In the present experiments, expression of mouse PrP in GFAP-HaPrP transgenic mice completely prevented clinical disease, and diminished HaPrPSc formation and propagation of hamster scrapie agent. Similar competitive or inhibitory effects between coexpressed PrP molecules of different species have been noted previously both in transgenic mice and in mouse neuroblastoma cell cultures.These results indicate, once more that agent propagation to high levels is not necessarily sufficient to cause pathology or clinical symptoms.
In the course of the disease in wild-type mice, PrPSc has been found to accumulate in astrocytes prior to the development of neuropathological changes (Diedrich et al., 1991), suggesting that PrPSc released into the extracellular compartment (Jeffrey et al., 1994) might contribute to pathogenesis by inherent toxicity. However, as shown recently , PrPSc was unable to elicit a neurotoxic effect when presented to PrP0/0 neural tissue; neurotoxicity only resulted when cells were PrP+/+ and thus able to produce PrPSc endogenously. Tg3/Prnp0/0 mice express HaPrPC in astrocytes but not in neurons, and therefore in these animals only astrocytes should be susceptible to direct pathological effects mediated by PrPSc.
However, because astrocytes are involved in maintaining the normal status of neurons, astrocyte dysfunction might result in neuronal abnormalities. Interestingly, onset and development of neuropathological changes in scrapie correlate with increased expression of a variety of cytokines. Such cytokines might be produced by astrocytes or other glial cells, and might play a key role in the pathological events of scrapie via indirect effects leading to neuronal dysfunction, analogous to that which has been seen following infection of glial cells by murine retroviruses which cause a spongiform pathology quite like scrapie.
Our findings that astrocytosis and spongiosis in the terminal phase of scrapie are quite similar in wild-type mice and in mice expressing PrP in either neurons or astrocytes only, suggest that a common final pathway, perhaps cytokine overproduction, contributes to the neuropathological picture in all these cases.
Glia 1997 Oct;21(2):253-257 Herms JW, Madlung A, Brown DR, Kretzschmar HAA synthetic peptide consisting of amino acid residues 106 to 126 of the human prion protein (PrPc) that forms fibrils in vitro is toxic to cultured neurons. We have previously shown that the neurotoxic effect of this peptide is related to microglia activation (Brown et al., 1996a). For closer insight into this process of activation, we investigated the effect of the peptide on the intracellular free Ca2+ concentration ([Ca2+]i) in cultured microglia using Fura-2. Cultured microglia from wild-type as well as from PrPc gene-ablated mice (Prn-p0/0) responded to exposure to PrP106-126 with an increase in intracellular free calcium within 30 min.
We observed two types of responses. Both in wild-type and Prn-p0/0 mice about half of the tested cells presented a small and often transient calcium increase after peptide application which was found to be independent of the extracellular calcium concentration. However, a further 33% of wild-type cells showed a strong and often permanent calcium increase depending on the extracellular calcium concentration, which was only rarely observed in Prn-p0/0 cells. To determine whether the response depended on the activation state of the microglia, we also examined LPS-treated activated microglia. The character of the calcium response remained unchanged, but significantly fewer cells responded. Our findings demonstrate the earliest reaction of microglia to a PrP fragment known to date.