Prion Disease: New Approaches to Therapy and Diagnosis
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The risk of blood-borne CJD
Structure-based drug design
Impaired prion replication in mice lacking follicular dendritic cells
N-methylation - a general strategy to prevent amyloid formation
Inhibition of huntingtin fibrillogenesis by Congo red etc.
Heterologous prion binding, inhibition of conversion, and species barriers
Accumulation of protease-resistant prion protein in 9x insertional mutants
Infectivity of Alzheimer seeding
Amyloid plaques found in aging salmon
Familial Danish dementia
Amyloid channel toxicity: prion, AD, islet amylin, poly-glutamine
Follow-up on heme, amphotericin therapy
Hay mites at it again

The risk of blood-borne CJD

Dev Biol Stand 2000;102:53-9
Brown P
With the increasing ability of modern medicine to transfer life-giving tissues and tissue products from one individual to another comes a parallel, often life-taking consequence of the unsuspected transfer of infectious passengers.

Creutzfeldt-Jakob disease (CJD) has been transmitted by contaminated stereotactic EEG electrodes, neurosurgical instruments, dura mater allografts, and cadaveric pituitary hormone therapy. Widespread concern has been voiced about the possible risks of transmitting CJD through the administration of blood or plasma products, although no such case has so far been identified by epidemiological studies of at-risk populations.

Recently completed experiments using hamsters and mice showed decreasing levels of infectivity in buffy coat, plasma, cryoprecipitate (the source of anti-haemophilic factor), and fraction I + II + III (the source of immune globulin). Preliminary results from further rodent experiments indicate that little or no infectivity is detectable in plasma during the pre-clinical phase of infection, that plasma infectivity is not reduced by either leukodepletion filtration or high speed centrifugation, and that about 5-10 times more infectivity is needed to transmit disease by the intravenous than intracerebral route.

Altogether, the data from these rodent studies, especially when considered together with results from similar studies underway in primates, should provide a rational foundation for policy decisions by government agencies, plasma product manufacturers, and blood anking communities.

Comment (webmaster): Further details on the Schmeer blood test were given at the 8 May 00 meeting in Miami. A video of the talk was made and will be available shortly. The test seems no closer than ever to validation and release, though it may have underlying merit. Other researchers cannot replicate results because a secret extraction step is used, even though this is surely patent-protected by now.

Differential expression of cellular prion protein on human blood and tonsil lymphocytes.

Haematologica 2000 May;85(5):475-480
Antoine N, Cesbron JY, Coumans B, Jolois O, Zorzi W, Heinen E
The expression of cellular prion protein (PrPc) on the surface of peripheral lymphocytes has been previously reported, but little is known about its expression on mphoid cells from secondary lymph organs. In this report, we compare the surface expression of PrPc on human blood lymphocytes and tonsil lymphocytes. This analysis was performed by cytometry on live lymphocytes isolated from healthy donors or from the tonsils of adults or children.

Human peripheral lymphocytes and tonsillar lymphoid cells, but not erythrocytes or granulocytes, express PrPc at their surfaces. Interestingly, we found significantly less PrPc on freshly isolated tonsil lymphocytes, both B and T, than on blood cells. Although tonsil cells bear less PrPc than circulating blood lymphocytes, they are able to express high quantities of PrPc on their surface when placed in culture. However, contrary to previous results, mitogen stimulation does not affect this expression on B- or T-cells.

We suggest that the PrPc expression by lymphocytes may be modified by interactions occurring during intratissular migration or during cell-to-cell contacts. Whether PrPc plays a role in intracellular communication at this location, as it does in the nervous system, remains an open question.

Ultrasensitive detection of pathological prion protein aggregates by dual- color scanning for intensely fluorescent targets

Proc. Natl. Acad. Sci. USA 2000 97(10): p. 5468-5473
J. Bieschke, A. Giese, W. Schulz-Schaeffer, I. Zerr, S. Poser, M. Eigen, and H. Kretzschmar
A definite diagnosis of prion diseases such as Creutzfeldt-Jakob disease (CJD) relies on the detection of pathological prion protein (PrPSc). However, no test for PrPSc in cerebrospinal fluid (CSF) has been available thus far. Based on a setup for confocal dual-color fluorescence correlation spectroscopy, a technique suitable for single molecule detection, we developed a highly sensitive detection method for PrPSc. Pathological prion protein aggregates were labeled by specific antibody probes tagged with fluorescent dyes, resulting in intensely fluorescent targets, which were measured by dual-color fluorescence intensity distribution analysis in a confocal scanning setup. In a diagnostic model system, PrPSc aggregates were detected down to a concentration of 2 pM PrPSc, corresponding to an aggregate concentration of approximately 2 fM, which was more than one order of magnitude more sensitive than Western blot analysis.

A PrPSc-specific signal could also be detected in a number of CSF samples from patients with CJD but not in control samples, providing the basis for a rapid and specific test for CJD and other prion diseases. Furthermore, this method could be adapted to the sensitive detection of other disease-associated amyloid aggregates such as in Alzheimer's disease.

Cultured cell sublines highly susceptible to prion infection

Journal of Virology, May 2000, p. 4377-4386, Vol. 74, No. 9
Patrick J. Bosque  and Stanley B. Prusiner
Cultured cell lines infected with prions produce an abnormal isoform of the prion protein (PrPSc). In order to derive cell lines producing sufficient quantities of PrPSc for most studies, it has been necessary to subclone infected cultures and select the subclones producing the largest amounts of PrPSc. Since postinfection cloning can introduce differences between infected and uninfected cell lines, we sought an approach to generate prion-infected cell lines that would avoid clonal artifacts.

Using an improved cell blot technique, which permits sensitive and rapid comparison of PrPSc levels in multiple independent cell cultures, we discovered marked heterogeneity with regard to prion susceptibility in tumor cell sublines. We exploited this heterogeneity to derive sublines which are highly susceptible to prion infection and used these cells to generate prion-infected lines without further subcloning. These infected sublines can be compared to the cognate uninfected cultures without interference from cloning artifacts.

We also used susceptible cell lines and our modified cell blot procedure to develop a sensitive and reproducible quantitative cell culture bioassay for prions. We found that the sublines were at least 100-fold more susceptible to strain RML prions than to strain ME7 prions. Comparisons between scrapie-susceptible and -resistant cell lines may reveal factors that modulate prion propagation.

Mimicking dominant negative inhibition of prion replication through structure-based drug design

May 23, 2000 Proc. Natl. Acad. Sci. USA, Vol. 97, Issue 11, 6073-6078
Véronique Perrier, Andrew C. Wallace,, Kiyotoshi Kaneko, Jiri Safar, Stanley B. Prusiner, and Fred E. Cohen
Comment (webmaster): This is really the first serious paper on finding a therapy for prion diseases; earlier work has investigated unmotivated 'magic bullets' not amenable to a systematic screening and refinement program. The approach here, though still at a preliminary stage, is very modern with its rational use of chemical libraries. Figure 1 describes five potential strategies for drug discovery suggested by their model:
1) block PrPC synthesis,
2) stabilize PrPC,
3) enhance PrPSc clearance,
4) interfere with binding of PrPC to PrPSc, and
5) prevent binding of protein X to PrPC.
The paper goes on too much about the mythical 'protein X' which the webmaster learned separately they have been unable to purify or for that matter even establish as a protein, supposedly because of low levels, making it more of a data 'interpreton' than a physical object at this point. At a minimum, it would be better called 'factor X' or "concept X" or nothing at all until it has been confirmed by a second lab and physically characterized.

Caution: compounds mentioned in the article are not ready for clinical use. People reading this article who are possibly impacted by CJD should not use them for self-medication. There are no therapeutic compounds suitable for human or animal use at this time.

Recent progress determining the structure of the host-encoded prion protein (PrPC) and the role of auxiliary molecules in prion replication permits a more rational approach in the development of therapeutic interventions. Our objective is to identify a new class of lead compounds that mimic the dominant negative PrPC mutants, which inhibit an abnormal isoform (PrPSc) formation. A computational search was conducted on the Available Chemicals Directory for molecules that mimic both the spatial orientation and basic polymorphism of PrP residues 168, 172, 215, and 219, which confer dominant negative inhibition.

The search revealed 1,000 potential candidates that were visually analyzed with respect to the structure of this four-residue epitope on PrPC. Sixty-three compounds were tested for inhibition of PrPSc formation in scrapie-infected mouse neuroblastoma cells (ScN2a).

Two compounds, Cp-60 (2-amino-6-[(2-aminophenyl)thio]-4-(2-furyl)pyridine-3,5-dicarbonitrile) and Cp-62 (N'1-({5-[(4,5-dichloro-1H-imidazol-1-yl)methyl]-2-furyl}carbonyl)-4 methoxybenzene-1-sulfonohydrazide), inhibited PrPSc formation in a dose-dependent manner and demonstrated low levels of toxicity.

A substructure search of the Available Chemicals Directory based on Cp-60 identified five related molecules, three of which exhibited activities comparable to Cp-60. Mimicking dominant negative inhibition in the design of drugs that inhibit prion replication may provide a more general approach to developing therapeutics for deleterious protein-protein interactions. ...

We present a structure-based approach to the identification of small heterocyclic molecules that were designed to mimic the dominant negative inhibition of prion replication by polymorphic variants of PrP. Although most structure-based drug design focuses on filling cavities on the surface of a molecule to inhibit an interaction or on optimizing the efficiency of small molecule whose detailed interactions with its molecular target are visualized crystallographically, we followed a distinctly different route. We sought to mimic the surface of a macromolecule by using a small molecule.

To the extent that our computational algorithm identifies appropriate mimetics of the PrPC epitope that specifies the dominant negative phenotype, PrPSc formation will be blocked. Our studies demonstrate that it is possible to identify plausible mimetics of a localized epitope on the surface of PrPC and that a subset of these molecules inhibits prion replication in cultured cells....

Our results show that Cp-60 is able to inhibit the formation of newly synthesized PrPSc and cure infected cells. However, additional bioassay studies will be required to confirm that prion infectivity has been eliminated. We also lack direct evidence that the mechanism of action of Cp-60 is indeed to block the PrPC/protein X interaction by mimicking the dominant negative epitope. Although protein X has yet to be isolated, derivatives of Cp-60 carrying a photoactivatable crosslinking moiety have been synthesized and observed to be more potent than the original compound. Thus derivatives of Cp-60 may provide a more direct route to the purification of protein X.

The mechanism of inhibition of PrPSc formation for Cp-60 seems to be radically different from that of Congo red. Cp-60 exhibits a classical dose-response curve (Fig. 4B), whereas low concentrations of Congo red increase the production of PrPSc approximately 3-fold and high concentrations block PrPSc formation (data not shown) (46). These observations are consistent with the direct binding of PrPSc to Congo red in a mode that initially stabilizes the molecular complex but ultimately occludes molecular surfaces that are required for PrPSc to act as a replication template.

Dominant-negative inhibition of prion formation diminished by deletions

Journal of Virology, May 2000, p. 4351-4360, Vol. 74, No. 9
Laurence Zulianello, Kiyotoshi Kaneko,..., Dong Han,  Fred E. Cohen, and Stanley B. Prusiner
Polymorphic basic residues near the C terminus of the prion protein (PrP) in humans and sheep appear to protect against prion disease. In heterozygotes, inhibition of prion formation appears to be dominant negative and has been simulated in cultured cells persistently infected with scrapie prions. The results of nuclear magnetic resonance and mutagenesis studies indicate that specific substitutions at the C-terminal residues 167, 171, 214, and 218 of PrPC act as dominant-negative, inhibitors of PrPSc formation (K. Kaneko et al., PNAS 94:10069-10074, 1997).

Trafficking of substituted PrPC to caveaola-like domains or rafts by the glycolipid anchor was required for the dominant-negative phenotype; interestingly, amino acid replacements at multiple sites were less effective than single-residue substitutions. To elucidate which domains of PrPC are responsible for dominant-negative inhibition of PrPSc formation, we analyzed whether N-terminally truncated PrP(Q218K) molecules exhibited dominant-negative effects in the conversion of full-length PrPC to PrPSc.

We found that the C-terminal domain of PrP is not sufficient to impede the conversion of the full-length PrPC molecule and that N-terminally truncated molecules (with residues 23 to 88 and 23 to 120 deleted) have reduced dominant-negative activity. Whether the N-terminal region of PrP acts by stabilizing the C-terminal domain of the molecule or by modulating the binding of PrPC to an auxiliary molecule that participates in PrPSc formation remains to be established.

Seeding of beta-amyloid by intracerebral infusion of Alzheimer Brain Extracts

Journal of Neuroscience, May 15, 2000,20(10):3606-3611
Michael D. Kane, William J. Lipinski,..., and Lary C. Walker
Comment (webmaster): This experiment would have been more impressive had purified protein, rather than neocortical homogenate, been infused. Note too the "non-AD brain with mild beta-amyloidosis" was also transmissive. The claim of 'seeding' is not fully established by their data (though may very well have occurred); perhaps higher levels of gamma secretase or other amyloid-limiting protease was the active factor. It is quite possible that many amyloidoses can be transmitted using the methods here. Given the incidence of Alzheimer plus "non-AD brain with mild beta-amyloidosis", there may be medical safety issues involving transplantation (eg, dura mater) and Alzheimer.

Many neurodegenerative diseases are associated with the abnormal sequestration of isease-specific proteins in the brain, but the events that initiate this process remain unclear. To determine whether the deposition of the beta-amyloid peptide (Abeta), a key pathological feature of Alzheimer's disease (AD), can be induced in vivo, we infused dilute supernatants of autopsy-derived neocortical homogenates from Alzheimer's patients unilaterally into the hippocampus and neocortex of 3-month-old beta-amyloid precursor protein (APP)-transgenic mice.

Up to 4 weeks after the infusion there was no Abeta-deposition in the brain; however, after 5 months, the AD-tissue-injected hemisphere of the transgenic mice had developed profuse Abeta-immunoreactive senile plaques and vascular deposits, some of which were birefringent with Congo Red.

There was limited deposition of diffuse Abeta also in the brains of APP-transgenic mice infused with tissue from an age-matched, non-AD brain with mild beta-amyloidosis, but none in mice receiving extract from a young control case. Abeta deposits also were not found in either vehicle-injected or uninjected transgenic mice or in any nontransgenic mice.

The results show that cerebral beta-amyloid can be seeded in vivo by a single inoculation of dilute AD brain extract, demonstrating a key pathogenic commonality between beta-amyloidosis and other neurodegenerative diseases involving abnormal protein polymerization. The paradigm can be used to clarify the conditions that initiate in vivo beta-amyloidogenesis in the brain and may yield a more authentic animal model of Alzheimer's disease and other neurodegenerative disorders.

Calcium channel toxicity: prion, AD, islet amylin

J Biol Chem 2000 May 5;275(19):14077-14083
Kawahara M, Kuroda Y, Arispe N, Rojas E
Comment (webmaster): The basic mystery in amyloidoses has always been the nature of the initial event in the chain of pathology, ie, why (or is) the amyloid itself toxic. There are now several papers with data supporting the idea of amyloid fibrils forming transmembrane pores, allowing ion leakage and subsequent cell death (due to energy depletion from pumping or too much of a particular ion). An alternative proposal sometimes put forward is that cross beta fosters formation of reactive oxidant species.

Both mechanisms have the required property of universality in that many unrelated proteins could be toxic through a common mechanism traceable to a universal cross beta architecture which does not involve particulars of side chain .

Alzheimer's beta-amyloid, human islet amylin, and prion protein fragment evoke intracellular free calcium elevations by a common mechanism

A growing number of reports suggest that elevated levels of extracellular Alzheimer's beta-amyloid protein alter the homeostasis of free [Ca(2+)](i) in different cell types of the mammalian brain. In line with these results, we have previously shown that AbetaP[1-40] forms cation-selective channels (Ca(2+) included) across artificial planar bilayers formed from acidic phospholipids and across excised membrane patches from immortalized hypothalamic GnRH neurons (GT1-7 cells), suggesting that the nonregulated Ca(2+)-influx through these spontaneously formed "amyloid channels" may provide a mechanism to explain its toxicity.

We have now found and report here that the application of AbetaP[1-40] to GT1-7 neurons consistently elevates [Ca(2+)](i) levels. We also found that human islet amylin and the prion protein fragment (PrP106-126), peptides that acquire beta-pleated sheet conformation in water solutions and have been reported to form ion channels across planar bilayer membranes, also increase cytosolic free calcium in GT1-7 neurons.

Searching for protective agents, we found that soluble cholesterol, known to decrease the fluidity of the cell membrane, inhibits AbetaP[1-40]-evoked [Ca(2+)](i) rise. These results suggest that unregulated Ca(2+) entry across amyloid channels may be a common mechanism causing cell death, not only in diseases of the third age, including Alzheimer's disease and type 2 diabetes mellitus, but also in prion-induced diseases.

Poly-L-Glutamine Forms Cation Channels: Relevance to the Pathogenesis of the Polyglutamine Diseases

Biophys J, June 2000, p. 2892-2899, Vol. 78, No. 6
Hiroshi Monoi, Shiroh Futaki, Shin-ichi Kugimiya, Hiroyuki Minakata, and Kazuo Yoshihara
We report that long-chain poly-L-glutamine forms cation-selective channels when incorporated into artificial planar lipid bilayer membranes. The channel was permeable to alkali cations and H+ ions and virtually impermeable to anions; the selectivity sequence based on the single-channel conductance was H+ Cs+ > K+ > Na+. The cation channel was characterized by long-lived open states (often lasting for several minutes to tens of minutes) interrupted by brief closings.

The appearance of the channel depended critically on the length of polyglutamine chains; ion channels were observed with 40-residue stretches, whereas no significant conductance changes were detected with 29-residue tracts. The channel-forming threshold length of poly-L-glutamine was thus between 29 and 40 residues. A molecular mechanics calculation suggests a µ-helix (Monoi, 1995. Biophys. J. 69:1130-1141) as a candidate molecular structure of the channel.

The channel-forming nature of long-chain poly-L-glutamine may provide a clue to the elucidation of the pathogenetic mechanism of the polyglutamine diseases, a group of inherited neurodegenerative disorders including Huntington's disease.

A decamer duplication in 3' region of BRI gene gives an amyloid peptide associated dementia in Danish kindred

PNAS 10.1073/pnas.080076097 April 18, 2000 
Ruben Vidal, Tamas Révész,.. Jorge Ghiso, and Blas Frangione 
Comment (webmaster): This is a very interesting followup paper to familial British dementia covered earlier on this site. It turns out that familial Danish dementia (FDD) is the 'same' disease but with a different amyloidogenic mutation, rather reminiscent of GSS, FFI, and CJD.

It seems that the C-terminus of the BRI gene has an intrinsic tendency towards amyloid formation after a furin proteolytic fragment has formed. However, this only manifests itself in extensional mutations affecting the stop codon. In FBD, the stop codon itself is altered, causing 12 aa read-thru to the next in-frame stop codon. In FDD, a 10bp replication slippage mutation leads also to a 12 residue extension to the next out-of-frame stop codon. In both cases, the normal 22 residue terminal fragment, when extended with completely unrelated peptides, becomes amyloidogenic and congo-red positive.

Highlights of article:

Familial Danish dementia (FDD), also known as heredopathia ophthalmo-oto-encephalica, is an autosomal dominant disorder characterized by cataracts, deafness, progressive ataxia, and dementia. Neuropathological findings include severe widespread cerebral amyloid angiopathy, hippocampal plaques, and neurofibrillary tangles, similar to Alzheimer's disease. N-terminal sequence analysis of isolated leptomeningeal amyloid fibrils revealed homology to ABri, the peptide originated by a point mutation at the stop codon of gene BRI in familial British dementia.

Molecular genetic analysis of the BRI gene [located on chromosome 13q14] in the Danish kindred showed a different defect, namely the presence of a 10-nt duplication (795-796insTTTAATTTGT) between codons 265 and 266, one codon before the normal stop codon 267. The decamer duplication mutation produces a frame-shift in the BRI sequence generating a larger-than-normal precursor protein [which now extends to the next in-frame stop codon, generating a precursor protein of 277 amino acids instead of 266 amino acids], of which the amyloid subunit (designated ADan) comprises the last 34 C-terminal amino acids. This DNA duplication produces the loss of the C-terminal serine (codon 266) causing a change in the reading frame. Both amyloids have identical N-terminal amino acid sequence (first 22 amino acids) and a completely different C-terminal sequence (last 12 amino acids).

This de novo-created amyloidogenic peptide, associated with a genetic defect in the Danish kindred, stresses the importance of amyloid formation as a causative factor in neurodegeneration and dementia. [Immunohistochemical studies using the Ab 5282 specifically labeled Congo red positive, parenchymal, and vascular amyloid lesions in addition to Congo red negative deposits in brain tissue of three FDD cases.]

Heredopathia ophthalmo-oto-encephalica or familial Danish dementia (FDD) was originally described by Strömgren and collaborators, who identified nine cases in three generations... The disease is characterized by the progressive development of cataracts and other ocular disorders (including ocular hemorrhages), hearing impairment, varying neurological symptoms, and dementia, usually associated with paranoid reactions and temporal disturbance of consciousness. Cataracts seem to be the first manifestation of the disease, starting before the age of 30, whereas impaired hearing normally develops 10-20 years later.

Cerebellar ataxia occurs shortly after the age of 40, followed by paranoid psychosis and dementia 10 years later. Most patients die in their fifth to sixth decade of life. Neuropathologically, the disease is characterized by (i) a uniform diffuse atrophy of all parts of the brain; (ii) a very severe chronic diffuse encephalopathy, mostly in the cerebellum, the cerebral cortex, and the white matter; and (iii) the presence of extremely thin and almost completely demyelinated cranial nerves.

A widespread amyloid angiopathy is present in the blood vessels of the cerebrum, choroid plexus, cerebellum, spinal cord, and retina. The presence of plaques and neurofibrillary tangles is the major histopathological finding in the hippocampus, whereas the cerebral white matter also shows some ischemic lesions. Herein, we report the isolation and biochemical characterization of a de novo-created amyloid protein and the identification of the genetic defect in the BRI gene that results in dementia in the Danish kindred.

The brain lesions in FDD are neuropathologically similar although not identical to the lesions observed in patients with FBD. In both conditions, there is severe amyloid angiopathy in the cerebrum and cerebellum with extensive hippocampal plaques including nonneuritic plaques, some resembling the cotton wool plaques of familial AD with PS1 delta9 mutation and neurofibrillary tangles. However, there are significant differences between the two diseases. In FDD the hippocampal deposits are mainly Congo-red negative, suggesting that the protein deposited is predominantly in a nonfibrillary structure. Furthermore, in the three cases with FDD, Abeta immunoreactivity was observed, mainly in a perivascular position.

Accumulation of protease-resistant prion protein in transgenic 9x mice

Proc. Natl. Acad. Sci. USA 2000 97(10): p. 5574-5579
Roberto Chiesa, Bettina Drisaldi, Elena Quaglio, Antonio Migheli, Pedro Piccardo, Bernardino Ghetti, and David A. Harris
Comment (webmaster): This is an excellent article with good experimental details and clear write-up. It provides the first useful mouse model of inherited prion disease, though admittedly a rare type -- 9 extra octapeptide repeats. [P102L mice, studied earlier, become ill but lack detectable Prp-res, making them less useful.] These transgenic mice open the door to many further studies of tissue distribution and turnover.

We have generated lines of transgenic mice that express a mutant prion protein (PrP) containing 14 octapeptide repeats whose human homologue is associated with an inherited prion dementia. These mice develop a neurological illness with prominent ataxia at 65 or 240 days of age, depending on whether the transgene array is, respectively, homozygous or hemizygous.

Starting from birth, mutant PrP is converted into a protease-resistant and detergent-insoluble form that resembles the scrapie isoform of PrP, and this form accumulates dramatically in many brain regions throughout the lifetime of the mice. As PrP accumulates, there is massive apoptosis of granule cells in the cerebellum....

We have recently developed a transgenic (Tg) mouse model of a familial prion disease by expressing the mouse PrP homologue of a nine-octapeptide insertional mutation (PG14) described in human patients (4). This insertion is the largest thus far identified in the PrP gene and is associated with a prion disease characterized by progressive dementia and ataxia, and by the presence of PrP-containing amyloid plaques in the cerebellum and basal ganglia (5-7).

Tg(PG14) mice develop a slowly progressive neurological disorder characterized clinically by ataxia and neuropathologically by PrP deposition in a synaptic-like pattern, gliosis, and loss of cerebellar granule cells. Moreover, PG14 PrP molecules expressed in the brains of the mice acquire the major biochemical properties of PrPSc, including partial resistance to proteinase K digestion, insolubility in nondenaturing detergents, and resistance to cleavage of the C-terminal glycolipid anchor by phospholipase. Thus, Tg(PG14) mice recapitulate several of the essential clinical, neuropathological, and biochemical features of inherited human prion diseases.

Although many studies of scrapie in rodents and other hosts have been carried out to understand the pathogenesis of infectiously acquired prion diseases, the absence of a suitable animal model has precluded similar analysis of the familial forms of these disorders. Several other lines of PrP transgenic mice have been described that spontaneously develop a neurological illness (8-11). However, only one of these expresses a mutant PrP (P101L) that is associated with a familial prion disease, and mice of this line do not produce detectable protease-resistant PrP in their brains.

Several fundamental questions about familial prion diseases therefore remain unexplored, such as the time course of PrPSc accumulation, the anatomical distribution of PrPSc production, and the relationship of PrPSc to the development of clinical symptoms and neuropathology. To address these issues, we undertook a prospective study of Tg(PG14) mice from birth through the terminal phase of their illness using a combined biochemical and histological approach. Our results provide important insights into the natural history and pathogenesis of familial prion diseases. ...

Although only limited pathological and biochemical information is available on human patients that carry the PG14 mutation, more extensive studies have been carried out on patients with a six-octapeptide PrP insertion (21), and in a number of these cases features quite similar to those seen in Tg(PG14) mice have been described, including nonamyloid PrP deposition, lack of spongiosis, and depletion of cerebellar granule cells. Thus, it is very likely that the results we have obtained from our analysis of Tg(PG14) mice will be applicable to human patients with inherited prion diseases.

Regardless of the exact correspondence between the phenotypes of our mice and those of human patients, however, our studies reveal several novel mechanistic insights into how a mutant form of PrP can cause neurodegeneration, including the role of the protease-resistant form of the protein in triggering the disease process, the cell death pathways that are activated, and the metabolism of the mutant protein in the brain.

Patients with familial prion diseases do not manifest symptoms until adulthood (2, 3), despite the fact that PrP is known to be expressed in the central nervous system beginning early in embryogenesis and to reach maximal levels soon after birth (22, 23). The question therefore arises of when during the lifetime of patients are mutant PrP molecules converted to the PrPSc state, and what role does PrPSc play in development of clinical illness? Is PrPSc produced beginning early in life or only in the adult? It has not been possible to address this question directly, since analysis of PrPSc in human subjects is of necessity restricted to a single time point at the terminal phase of the illness.

Our analysis of Tg(PG14) mice reveals that molecules with the biochemical properties of PrPSc accumulate throughout the life of the animal, but that neuropathological lesions and clinical disease do not ensue until a critical threshold level has been reached. In both Tg(PG14+/+) and Tg(PG14+/) mice, detergent-insoluble and protease-resistant PrP are already detectable within the first week of life, long before clinical symptoms commence (at 65 and 240 days of age, respectively, in the two genotypes), and before the initiation of astrocytosis and granule cell apoptosis (at 30 days in both genotypes).

PrPSc then accumulates in the brain as the mice age, reaching levels in terminally ill animals that are 20- to 80-fold higher than in newborn mice. This dramatic elevation of PrPSc is correlated with a marked astrocytic reaction and with progressive granule cell loss in the cerebellum. Of note, PrPSc increases more rapidly and reaches higher levels in Tg(PG14+/+) than in Tg(PG14+/) mice (Fig. 2 C and D), correlating with the faster disease progression in homozygous animals.

Taken together, these results argue strongly that PrPSc plays a primary pathogenic role in familial prion diseases, as it does in infectious forms, and is the causative factor in development of neuropathology and clinical symptoms. Our data also indicate that neurons at all stages of brain maturation possess the cellular and molecular machinery necessary for converting mutant PrP to a PrPSc-like state, and that this capability is not restricted to the adult nervous system.

Our results suggest that PG14 PrP accumulates in the brain because it turns over more slowly or is cleared less efficiently than WT PrP. The total amount of mutant PrP in the brains of Tg(PG14) mice increases ~10-fold during the life of the animal, whereas the amount of WT PrP in Tg(WT) and nontransgenic CD1 mice changes by <2-fold. Since levels of the mRNAs encoding mutant and WT PrP remain relatively constant after birth, the rates of synthesis of the two proteins are likely to be similar, assuming the absence of differential translational efficiency.

Whether PG14 PrP is degraded more slowly within cells or whether it accumulates in a more stable form in the extracellular space of the brain remains to be determined. We have found that PG14 PrP has a slower metabolic half-life than WT PrP when expressed in transfected Chinese hamster ovary cells (24), but whether the same is true when the protein is expressed in neurons is not known.

Familial prion diseases exhibit considerable variability in their clinical and neuropathological presentation. A hypothesis to explain the anatomical specificity of each mutation is that neurons from different parts of the brain metabolize mutant PrPs in different ways, and that some populations of neurons convert particular mutant PrPs to the PrPSc state much more efficiently than other populations of neurons.

Our analysis of Tg(PG14) mice indicates that this is not likely to be the case for the PG14 mutation. We find that PrPSc is widely distributed throughout the brains of both presymptomatic and terminally ill animals, as demonstrated by regional measurements of the amount of detergent-insoluble and protease-resistant PrP and by histoblotting of cryostat sections. Our results suggest that neurons in most parts of the brain are capable of converting PG14 PrP to a PrPSc-like state with similar efficiency. This conclusion is substantiated by studies of PrPSc formation in primary cultures of neurons derived from different brain regions of Tg(PG14) mice (B.D. and D.A.H., unpublished data).

Moreover, the ability to produce PrPSc-like molecules does not seem to be restricted to neurons, since many peripheral tissues in Tg(PG14) mice also contain a protease-resistant and detergent-insoluble form of the mutant protein (R. C. and D. A. H., unpublished data). In addition, PG14 PrP acquires PrPSc properties in transfected Chinese hamster ovary cells in culture (24-26). The conclusion that emerges from these data is that the PG14 mutation structurally alters the protein in such a way that promotes conversion to the PrPSc state regardless of the cellular context. Other pathogenic mutations may operate in a more cell-specific fashion, for example, the FFI mutation which produces a more selective pattern of PrPSc accumulation (27).

The widespread anatomical distribution of PrPSc seen by Western blotting and histoblotting contrasts with the more restricted localization of punctate PrP deposits visible by immunohistochemical staining. These deposits are present mainly in the cerebellum, hippocampus, and olfactory bulb. This discrepancy could be attributable to anatomical variations in the physical state of PrPSc. For example, if PrP in the cerebellum and hippocampus was more aggregated than in other regions of the brain, then the protein there might have a greater resistance to the denaturing treatments (hydrolytic autoclaving and incubation with guanidine thiocyanate) applied before immunohistochemical staining....

Interactions between heterologous forms of prion protein: Binding, inhibition of conversion, and species barriers

Proc. Natl. Acad. Sci. USA, 10.1073/pnas.110523897
Motohiro Horiuchi, Suzette A. Priola, Joëlle Chabry, and Byron Caughey
Comment (webmaster): This article continues a program of dissecting out stages of the conversion of normal prion protein to relative protease K resistance. Basically, an incoming seed fibril can recruit a normal monomer, but that binding step is distinct from subsequent conversion [fibril growth] and may even be interfering in the heterologous case [primary sequence mismatch due to species differance or internal alleles]. The authors calculate 25 prion monomers are needed in cross-beta form to give one crystal growth point. Thus it is epitaxial growth that forms a key rate-limiting step in cross-species infectivity. Example: an incoming bovine prion seed adds human monomers and gives rise to an in situ human seed that is far more effective in recruitment (as it is now homogeneous).

Nucleated polymerization-based models for binding, conversion (Upper), and interference (Lower) phenomena. Single-site (A) and two-binding-site (B) pathways are shown. In the single-site model (Upper, A), rapid binding of PrP-sen (open triangles) is followed by a slower wave of conformational conversion of bound PrP-sen molecules to PrP-res (squares). The inclusion of nonconvertible PrP-sen (black triangles) among convertible PrP-sen molecules prevents propagation of the conversion through the bound PrP-sen molecules (Lower, A). In the two-site models (B), binding of PrP-sen can occur either at the conversion-inducing site (shown at the end of the PrP-res polymer) or at a nonconverting site (e.g., on the sides of the polymer). Interference with conversion (Lower, B) could occur by blockade of the conversion site by nonconvertible PrP-sen without blocking binding of either type of PrP-sen to the nonconverting sites. PK designates a proteinase K digestion step wherein PrP-sen is completely digested and the N-terminal octapeptide repeat domain (residues 23-90, the wavy lines in the PrP-sen and PrP-res structures) are removed from PrP-res.

Highlights of the article:

"The self-induced formation of the disease-associated, protease-resistant prion protein (PrP-res) from the normal protease-sensitive isoform (PrP-sen) appears to be a key event in the pathogenesis of transmissible spongiform encephalopathies. The amino acid sequence specificity of PrP-res formation correlates with, and may account for, the species specificity in transmission of transmissible spongiform encephalopathy agents in vivo.

To analyze the mechanism controlling the sequence specificity of PrP-res formation, we compared the binding of PrP-sen to PrP-res with its subsequent acquisition of protease resistance by using cell-free systems consisting of heterologous versus homologous mouse and hamster PrP isoforms. Our studies showed that heterologous PrP-sen can bind to PrP-res with little conversion to the protease-resistant state and, in doing so, can interfere with the conversion of homologous PrP-sen. The interference occurred with molar ratios of homologous to heterologous PrP-sen molecules as low as 1:1.

The interference was due primarily to the inhibition of conversion, but not the binding, of the homologous PrP-sen to PrP-res. The results provide evidence that the sequence specificity of PrP-res formation in this model is determined more by the conversion to protease resistance than by the initial binding step. These findings also imply that after the initial binding, further intermolecular interactions between PrP-sen and PrP-res are required to complete the process of conversion to the protease-resistant state ....

In cell-free reactions in which PrP-res induces the conversion of PrP-sen to the protease-resistant form, two steps have been segregated kinetically; first, the binding of PrP-sen to PrP-res, and second, its acquisition of protease resistance (21, 22). This observation raises the question of which step requires amino acid sequence compatibility between PrP-sen and PrP-res. To address this question, we have quantitatively compared the initial binding and overall cell-free conversion reactions between mouse and hamster PrP isoforms. Binding of heterologous PrP-sen to PrP-res was observed with little conversion to the protease-resistant state. Furthermore, such binding of heterologous PrP-sen interfered with the PrP-res-induced conversion of homologous PrP-sen. These results provide insight not only into the molecular control of species specificity but also the basic mechanism of PrP-res formation itself. ...

The binding between PrP-sen and PrP-res is highly selective with respect to other proteins (22); however, as we have shown here, binding between heterologous PrP isoforms can occur readily. Interestingly, the subsequent step in which bound PrP-sen acquires protease resistance depends more on PrP sequence homology and may not occur between heterologous PrP species. Thus, with mouse and hamster isoforms of PrP at least, it is the acquisition of protease resistance by PrP-sen rather than the initial binding of PrP-sen to PrP-res that primarily determines the sequence specificity of PrP-res formation. ...

Taken together, these findings suggest the following sequence of events in the conversion process: Binding occurs initially through one or more of these surfaces independent of possible mismatches at residue 138/139 or other residues. The subsequent conversion to protease-resistant PrP, however, requires further, more specific, intermolecular interactions and/or conformational changes that are strongly influenced by residues 138/139, 154/155, and/or 169/170. Indeed, this was suggested previously to be the case for residue 138/139 because, in PrP-sen, it is hydrophobic, internally disposed, and unlikely to take part in initial intermolecular interactions with PrP-res.

Stoichiometry of Conversion Sites per Unit of PrP-res. We have also shown here that the formation of protease-resistant 35S-PrP in homologous reactions can be partially blocked by heterologous PrP-sen even when the latter is at ratios of 1:1 to the homologous 35S-PrP-sen and 1:50 to the PrP-res seed. This result suggests the following:

(i) the heterologous, nonconvertible PrP-sen is able to compete effectively with the homologous PrP-sen for the conversion-inducing binding site;

(ii) the ability of PrP-res to induce conversion of homologous PrP-sen is terminated once it is bound by conversion-incompetent heterologous PrP-sen (Fig. 6); and

(iii) there are far fewer conversion-inducing sites than PrP-res molecules in the average PrP-res polymer under these conditions.

The 50% inhibition of conversion that was observed with a 1:50 ratio of heterologous PrP-sen to PrP-res suggests that conversion-inducing binding sites for PrP-sen on PrP-res were roughly 50% occupied by the interfering PrP-sen. Thus, the number of conversion-inducing sites on the PrP-res polymers can be estimated to have been a maximum of 1 per 25 PrP-res molecules.

Impaired prion replication in spleens of mice lacking functional follicular dendritic cells

Science 2000 288(5469): p. 1257-1259
 Fabio Montrasio, Rico Frigg, Markus Glatzel, Michael A. Klein,   Fabienne Mackay, Adriano Aguzzi, and Charles Weissmann
Comment (webmaster): This is another substantial article in a series of sophisticated studies of the role of the immune system in early stages of TSE infections. Issues raised by Collinge in regards to earlier work have presumbably been addressed. The center of attention has remained follicular dendritic cells. The article is very compressed due to space limitations imposed by the journal and is not amenable to a brief summary here.

It is important to note that exposure to BSE for many people occurred 10-15 years ago; infectivity could have long since passed into the brain, mooting subsequent events in the immune system. Compounds mentioned in the article such as LTbetaR-Ig are not shown suitable for clinical use for CJD in humans and could have dangerous side effects.

"Replication of prions in the spleen and prion transfer from the periphery to the central nervous system (neuroinvasion) is impaired in certain forms of murine immunodeficiency (1-4), the presence of mature B lymphocytes being essential (5). In spleens of wild-type mice inoculated intraperitoneally (i.p.), infectivity is associated with B and T lymphocytes as well as with the stroma, but not with the pulp-derived non-B, non-T cell fraction or with circulating lymphocytes (6). Infectivity in the stroma is thought to reside in radiation-resistant, prion protein (PrP)-expressing postmitotic cells (7-9). A prime candidate is the FDC, because the pathological isoform of the prion protein, PrPSc, colocalizes with FDCs (10-12) and because chimeric mice harboring PrP-expressing but not PrP-deficient FDCs propagate prions after i.p. inoculation (9). Although PrP expression is essential for sustaining prion replication, B lymphocytes that lack PrP restore prion accumulation in spleen and neuroinvasion in severe combined immunodeficient (SCID), RAG-1/, and µMT mice (13), indicating a requirement for cells dependent on B cells or their products, such as mature FDCs....

Our results provide further evidence that FDCs are essential for accumulation of PrPSc and infectivity in the spleen and that they contribute, directly or indirectly, to neuroinvasion, complementing earlier conclusions based on immunocytochemical and genetic approaches. To what extent, if any, the long-term effects of LTR-Ig, such as the reduction of dendritic cell numbers ) or marginal zone macrophages , affect scrapie pathogenesis in the spleen has not been assessed. The requirement for B cells for prion replication in the spleen and efficient neuroinvasion is readily explained by their essential role in the maturation of FDCs. PrP knockout mice expressing PrP only in B cells do not sustain prion replication , suggesting that prions associated with splenic B cells may be acquired from FDCs. The delay in neuroinvasion caused by LTR-Ig may be due either to the fact that prion propagation in the spleen was interrupted for some 8 weeks or that a pathway bypassing the lymphoreticular system altogether was used . Because nvCJD affects the lymphoreticular system before the appearance of clinical symptoms, one might speculate that early diagnosis and long-term treatment with LTR-Ig could retard progression of the disease."

Tumor Necrosis Factor Alpha-Deficient, but Not Interleukin-6-Deficient, Mice Resist Peripheral Infection with Scrapie

Journal of Virology, April 2000, p. 3338-3344, Vol. 74, No. 7
Neil A. Mabbott, Alun Williams,... Giorgos Kollias, and Moira E. Bruce
In most peripheral infections of rodents and sheep with scrapie, infectivity is found first in lymphoid tissues and later in the central nervous system (CNS). Cells within the germinal centers (GCs) of the spleen and lymph nodes are important sites of extraneural replication, from which infection is likely to spread to the CNS along peripheral nerves.

Here, using immunodeficient mice, we investigate the identity of the cells in the spleen that are important for disease propagation. Despite possessing functional T and B lymphocytes, tumor necrosis factor alpha-deficient (TNF-/) mice lack GCs and follicular dendritic cell (FDC) networks in lymphoid tissues. In contrast, lymphoid tissues of interleukin-6-deficient (IL-6/) mice possess FDC networks but have impaired GCs. When the CNSs of TNF-/, IL-6/, and wild-type mice were directly challenged with the ME7 scrapie strain, 100% of the mice were susceptible, developing disease after closely similar incubation periods.

However, when challenged peripherally (intraperitoneally), most TNF-/ mice failed to develop scrapie up to 503 days postinjection. All wild-type and IL-6/ mice succumbed to disease approximately 300 days after the peripheral challenge. High levels of scrapie infection and the disease-specific isomer of the prion protein, PrPSc, were detectable in spleens from challenged wild-type and IL-6/ mice but not from TNF-/ mice. Histopathological analysis of spleen tissue demonstrated heavy PrP accumulations in direct association with FDCs in challenged wild-type and IL-6/ mice. No PrPSc accumulation was detected in spleens from TNF-/ mice.

We conclude that, for the ME7 scrapie strain, mature FDCs are critical for replication in lymphoid tissues and that in their absence, neuroinvasion following peripheral challenge is impaired.

Inhibition of toxicity in the beta-amyloid peptide fragment (25-35) using N-methylated derivatives - A general strategy to prevent amyloid formation

J. Biol. Chem, 10.1074/jbc.M003554200
Eleri Hughes, Ron M Burke, and Andrew J Doig 
Beta (25-35) is a synthetic derivative of beta-amyloid, the peptide that is believed to cause Alzheimer. As it is highly toxic and forms fibrillar aggregates typical of beta-amyloid, it is suitable as a model for testing inhibitors of aggregation and toxicity. We demonstrate that N-methylated derivatives of (25-35), which in isolation are soluble and non-toxic, can prevent the aggregation and inhibit the resulting toxicity of the wild type peptide. N-methylation can block hydrogen bonding on the outer edge of the assembling amyloid. The peptides are assayed by Congo red and Thioflavin T binding, electron microscopy and an MTT toxicity assay on PC12 cells.

One peptide (Gly 25 N-methylated) has similar properties to wild-type, while five have varying effects on prefolded fibrils and fibril assembly. In particular, (25-35) with Gly33 N-methylated is able to completely prevent fibril assembly and reduces the toxicity of prefolded amyloid. With Leu34 N-methylated the fibril morphology is altered and toxicity reduced. We suggest that the use of N-methylated derivatives of amyloidogenic peptides and proteins could provide a general solution to the problem of amyloid deposition and toxicity.

Inhibition of huntingtin fibrillogenesis by specific antibodies and small molecules: Implications for Huntington's disease therapy

PNAS 10.1073/pnas.110138997
Volker Heiser, Eberhard Scherzinger, Annett Boeddrich, Eckhard Nordhoff, Rudi Lurz, Nancy Schugardt, Hans Lehrach, and Erich E.
The accumulation of insoluble protein aggregates in intra and perinuclear inclusions is a hallmark of Huntington's disease (HD) and related glutamine-repeat disorders. A central question is whether protein aggregation plays a direct role in the pathogenesis of these neurodegenerative diseases. Here we show by using a filter retardation assay that the mAb 1C2, which specifically recognizes the elongated polyglutamine (polyQ) stretch in huntingtin, and the chemical compounds Congo red, thioflavine S, chrysamine G, and Direct fast yellow inhibit HD exon 1 protein aggregation in a dose-dependent manner. Cell culture studies revealed that the Congo red dye at micromolar concentrations reduced the extent of HD exon 1 aggregation in transiently transfected COS cells.

On the other hand, potential inhibitors of amyloid- formation such as thioflavine T, gossypol, melatonin, and rifampicin had little or no inhibitory effect on huntingtin aggregation in vitro. Together, these findings contribute to a better understanding of the mechanism of huntingtin fibrillogenesis in vitro and provide the basis for the development of new huntingtin aggregation inhibitors that may be effective in treating HD.

Huntington's disease (HD) is a progressive neurodegenerative disorder with a generally midlife age of onset and a duration of 15-20 years. The disease is characterized by personality changes, motor impairment, and subcortical dementia (1). It is associated with selective neuronal cell death in the cortex and striatum (2). The mutation that causes HD is a CAG/polyglutamine (polyQ) repeat expansion in the first exon of the HD gene encoding the huntingtin protein. The molecular mechanisms responsible for delayed onset, selective pattern of neuropathology, and cell death observed in HD are unknown. However, insoluble huntingtin protein aggregates have been detected in an in vitro model system (3), as well as in transgenic animals (4), fly models (5), cell culture systems (6), and brains of HD patients (7). PolyQ-containing protein aggregates also have been found in related glutamine-repeat disorders such as dentatorubral pallidoluysian atrophy, spinal bulbar muscular atrophy, and the spinocerebellar ataxias type 1, 2, 3, and 7, suggesting that all of these neurodegenerative diseases are caused by deposition of toxic protein aggregates.

Although the causal relationship between aggregate formation and disease has not been proven, genetic, neuropathological, and biochemical evidence indicate that formation of insoluble protein aggregates plays an important role in the cellular distortions underlying HD and the related glutamine-repeat disorders. Recently, Ona et al. (8) have demonstrated that expression of a dominant-negative caspase-1 mutant slows down aggregate formation of the HD exon 1 protein as well as disease progression in transgenic mice.

Furthermore, evidence has been presented that certain components of the proteasome, transcription factors, chaperones, and caspases, which normally are essential for cell viability, are recruited into polyQ-containing aggregates (9, 10). Accumulation of caspase-8 into insoluble protein aggregates, for example, is required for induction of cell death in primary rat neurons, whereas prevention of caspase-8 recruitment into aggregates blocks polyQ-induced cell death (11). Taken together these results suggest that formation of insoluble polyQ-containing protein aggregates is important both for the initiation and progression of these late-onset neurodegenerative disorders. ...

Our study demonstrates that the mAb 1C2 as well as the chemical compounds Congo red, thioflavine S, Direct fast yellow, and chrysamine G are capable of preventing huntingtin aggregation in vitro, at least partially. Although the molecular mechanism by which the 1C2 antibody and the various compounds inhibit huntingtin fibrillogenesis is unknown, we suggest that the antibody has a chaperone-like activity and blocks aggregation by stabilizing the native conformation of the elongated polyQ tract, whereas Congo red and its derivatives, which are known to selectively bind to -sheet structures (27) may slow down huntingtin aggregation by interfering with nucleus formation and/or growth of the fibrils.

Perutz proposed that elongated polyQ chains beyond a critical length (40+ glutamines) may lead to a phase change from random coils to hydrogen-bonded hairpins that assemble into -sheet structures. The finding that the 1C2 antibody, which does recognize elongated polyQ chains in soluble proteins but not in SDS-insoluble aggregates, inhibits huntingtin aggregation supports this hypothesis and suggests that other molecules that are capable of stabilizing the native conformation of an elongated polyQ tract should be effective in inhibiting huntingtin aggregation.

However, experiments to study antigen/antibody binding and its effect on the self-assembly of polyQ aggregates in more detail are necessary. A major drawback of the in vivo use of antibodies or peptides is that they may be degraded by proteases and that they have a poor blood-brain permeability. In addition, they may not enter the neuronal cells, where the aggregates are formed. Therefore, in future studies small molecules or chemically modified peptides should be screened that are protease resistant, have a reasonable brain permeability, and stabilize the conformation of the polyQ tract in the mutant huntingtin protein.

Our results suggests that lipophilic derivatives of Congo red such as chrysamine G and Direct fast yellow that specifically bind to amyloid-like -sheet structures have a considerable therapeutic potential. Recently, we proposed that inhibition of nucleus formation is a feasible therapeutic strategy against HD and the related glutamine-repeat disorders (15). Our findings support this hypothesis because treatment of transiently transfected COS cells with Congo red significantly reduced the formation of insoluble huntingtin aggregates in vivo. Furthermore, after overnight incubation of trypsin-treated GST-HD51 fusion protein soluble HD51 protein was detected only by immunoblotting or MS when Congo red was present during incubation (Figs. 3 and 4). These results suggest that Congo red interferes with nucleus formation and thereby significantly elongates the lag time of the aggregation process.

However, more extensive research, including toxicological studies will be necessary to determine whether the more lipophilic chemical compounds chrysamine G and Direct fast yellow can be used as huntingtin aggregation inhibitors in vivo. In addition, a large-scale screening for the identification of new huntingtin aggregation inhibitors by using an automated membrane filter retardation assay is in progress.

Although it has not been formally proven whether the formation of ordered huntingtin aggregates is the cause of HD, we propose that inhibition of huntingtin fibrillogenesis by small molecules is a very attractive therapeutic strategy. Drugs that selectively bind to elongated polyQ sequences should delay the onset and progression of HD and related glutamine-repeat disorders. Moreover, they also may be useful for treatment of other neurodegenerative disorders caused by aberrant protein folding such as Alzheimer's disease, Parkinson's disease, or the prion diseases.

Characteristics of scrapie isolates derived from hay mites.

Journal of NeuroVirology (2000) 6, 137 - 144.
 Carp RI, Meeker HC, Rubenstein R, Sigurdarson S, Papini M, Kascsak RJ, Kozlowski PB, Wisniewski HM
Previous epidemiological evidence suggested that in some instances a vector and/or reservoir is involved in the occurrence and spread of transmissible spongiform encephalopathies (TSEs). In a preliminary study, hay mite preparations from five Icelandic farms with a history of scrapie were injected into mice, and some of these mice became sick after long incubation periods. To confirm that the disease was scrapie, subsequent passages in mice were performed. In addition, the characteristics of the disease process in these passages were assessed and the results compared to those findings with standard scrapie strains.

As expected for scrapie, subsequent passages in the same host led to shortened incubation periods compared to those in primary isolate mice, and all mice had spongiform changes in brain. Results were similar for three of four isolates with regard to clinical manifestations, the incubation periods in mice of the three scrapie incubation-period genotypes (s7s7, s7p7, p7p7), and the PrPSc Western blot (WB) pattern. The characteristics of the fourth isolate were markedly different from the other three isolates with regard to these parameters. Comparison of the characteristics of standard mouse-adapted scrapie strains and the four isolates revealed differences; these differences were particularly pronounced for the fourth isolate.

Porphyrin and phthalocyanine antiscrapie compounds

Science 2000 Feb 25;287(5457):1503-6 
Priola SA, Raines A, Caughey WS
The likelihood that a bovine form of TSE has crossed species barriers and infected humans underscores the urgent need to identify anti-TSE drugs. Certain cyclic tetrapyrroles (porphyrins and phthalocyanines) have recently been shown to inhibit the in vitro formation of PrP-res, a protease-resistant protein critical for TSE pathogenesis. We now report that treatment of TSE-infected animals with three such compounds increased survival time from 50 to 300%. The significant inhibition of TSE disease by structurally dissimilar tetrapyrroles identifies these compounds as anti-TSE drugs.

Studies have shown that there is a close correlation between compounds that inhibit PrP-res formation in vitro and compounds that inhibit TSE disease (4). Therefore, PrP-res is an obvious target for therapeutic intervention. Certain cyclic tetrapyrroles, a class of compounds that includes biologically important hemes and chlorophylls, bind strongly and selectively to proteins and effect changes in protein conformation. Thus, these compounds were good candidates for

inhibitors of PrP-res formation and TSE disease. We have recently demonstrated that the cyclic tetrapyrroles PcTS (phthalocyanine tetrasulfonate), TMPP-Fe3+ [meso-tetra(4- N-methylpyridyl)porphine iron(III)], and DPG2-Fe3+ [deuteroporphyrin IX 2,4-bis(ethylene glycol) iron(III)] (Fig. 1) all strongly inhibited PrP-res formation in vitro (7).

To determine if they could also inhibit TSE disease in vivo, we infected transgenic mice overexpressing hamster PrP-sen (Tg7) (8) intraperitoneally (IP) with a high dose of hamster 263K scrapie. Starting on the day of infection [0 days after infection (dpi)], animals were injected IP three times a week over 4 weeks with one of the three compounds, for a total of 12 treatments (9, 10). Every compound tested significantly delayed disease when compared with untreated controls (Fig. 2A). Treatment with DPG2-Fe3+, which of the three compounds tested was the least effective inhibitor of PrP-res formation in vitro (7), increased mean survival time by 37 days, whereas treatment with the stronger inhibitor TMPP-Fe3+ increased mean survival time by 90 days (Table 1).

PcTS, the strongest inhibitor of PrP-res in vitro, was also the most effective compound in vivo, with over 50% of the animals surviving an average of 135 days longer than untreated controls (Fig. 2A, Table 2). Similar levels of PrP-res were detected in the brains of terminally ill treated and untreated animals (11). The data demonstrated that three tetrapyrroles, each of a structurally different type, strongly inhibited TSE disease, showing that cyclic tetrapyrroles can act as anti-TSE agents. Animals were infected IP with 0.05 ml of a 1:10 dilution of a stock 10% brain homogenate of hamster 263K scrapie. The stock had an intracranial median lethal dose of 1 x 10exp10/ml.

Amphotericin B Inhibits the Generation of the Scrapie Isoform of the Prion Protein in Infected Cultures

Journal of Virology, April 2000, p. 3135-3140, Vol. 74, No. 7 $04.00
Alain Mangé, Noriyuki Nishida, ..Danielle Casanova, and Sylvain Lehmann
...Polyene antibiotics such as amphotericin B have been shown to delay the accumulation of PrPSc and to increase the incubation time of the disease after experimental transmission in laboratory animals. Unlike for Congo red and sulfated polyanions, no effect of amphotericin B has been observed in infected cultures. We show here for the first time that amphotericin B can inhibit PrPSc generation in scrapie-infected GT1-7 and N2a cells. Its activity seems to be related to a modification of the properties of detergent-resistant microdomains. These results provide new insights into the mechanism of action of amphotericin B and confirm the usefulness of infected cultures in the therapeutic research of transmissible spongiform encephalopathies.

Comment (webmaster): Neither the first article nor earlier ones provides any underlying rationale for specific binding of heme compounds. Prion protein does have a good RMS fit to 3 helices of hemoglobin but the variety of compounds tested are chemically inconsistent with specific binding. Since all compounds tested worked, control compounds are needed that don't work (beyond solvent blanks). There is little interest in therapeutic compounds that must be injected on the day of exposure -- in England 15 years have gone by and the disease is likely entrenched behind the blood-brain barrier.

Both articles are typical of TSE therapy research to date: a pet compound, no systematic screening of chemical libraries, no involvement of pharmaceutical companies with actual experience in drug development, no overall clinical strategy, no coordination with research in other amyloidoses. It is all reminiscent of the 'magic bullet' approach to cancer formulated in 1908. In some ways, progress is less in the specific compounds than in more powerful methodologies for screening.

It is difficult to envision an untested neuroactive compound being injected prophalacticly into 60 million exposed preclinical, indeed undiagnosed, individuals with no control group or long term studies of side effects and toxicity. Even familiar drugs could be come unsafe if the blood/brain barrier must be breached. So it is not just a question whether a blood test can detect the disease prior to damage (as there can be no interest in stabilizing a patient after disease onset).

Congo red is the probably the most interesting class of compounds in that it binds specifically to universal cross-beta fibrils in a known manner and so is not tied to primary sequence nor normal protein conformation. In many amyloidoses, intervention must come at the level of already-formed fibril. H4>Screening Congo Red and its analogues for their ability to prevent the formation of PrP-res in scrapie-infected cells

H. Rudyk, S. Vasiljevic, R. M. Hennion, C. R. Birkett, J. Hope and I. H. Gilbert 
J Gen Virol 2000 April 81: 1155-1164 

Novel therapeutic uses for porphyrins and phthalocyanines in the transmissible spongiform encephalopathies.

Curr Opin Microbiol 1999 Oct;2(5):563-6 
Priola SA, Caughey B, Caughey WS

Inhibition of protease-resistant prion protein formation by porphyrins and phthalocyanines.

PNAS 1998 Oct 13;95(21):12117-22
Caughey WS, Raymond LD, Horiuchi M, Caughey B
A central aspect of pathogenesis in the transmissible spongiform encephalopathies or prion diseases is the conversion of normal protease-sensitive prion protein (PrP-sen) to the abnormal protease-resistant form, PrP-res. Here we identify porphyrins and phthalocyanines as inhibitors of PrP-res accumulation. The most potent of these tetrapyrroles had IC50 values of 0.5-1 microM in scrapie-infected mouse neuroblastoma (ScNB) cell cultures. Inhibition was observed without effects on protein biosynthesis in general or PrP-sen biosynthesis in particular. Tetrapyrroles also inhibited PrP-res formation in a cell-free reaction composed predominantly of hamster PrP-res and PrP-sen. Inhibitors were found among phthalocyanines, deuteroporphyrins IX, and meso-substituted porphines; examples included compounds containing anionic, neutral protic, and cationic peripheral substituents and various metals.

We conclude that certain tetrapyrroles specifically inhibit the conversion of PrP-sen to PrP-res without apparent cytotoxic effects. The inhibition observed in the cell-free conversion reaction suggests that the mechanism involved direct interactions of the tetrapyrrole with PrP-res and/or PrP-sen. These findings introduce a new class of inhibitors of PrP-res formation that represents a potential source of therapeutic agents for transmissible spongiform encephalopathies.

Salmon ``clock'' may answer human aging queries

Mar 09, 2000 Reuters
Salmon with sticky deposits in their brains may offer clues to treating or even preventing Alzheimer's disease in humans, researchers said on Wednesday. A team at the University of Colorado at Boulder found that salmon develop deposits known as amyloid plaques in their brains that look much like the plaques that characterize Alzheimer's.

Comparisons of brain tissue from salmon and from a human who died with Alzheimer's disease showed they were ``very similar if not identical,'' Tammy Maldonado, who worked on the study, said in a statement. The researchers said stress hormones set off an ``alarm clock'' that starts the quick and relentless deterioration and death of salmon just after they spawn, and that a similar process may be involved in human Alzheimer's.

But Maldonado and colleagues reported in Friday's issue of the journal Brain Research that parts of the fishes' brains continued to work well despite deposits of the amyloid plaques. ``Instead of being murderers, amyloid plaques may be allies of the brain during times of stress and trauma,'' said Richard Jones, a professor emeritus in the environmental, population and organismic biology department, who also worked on the study.

``Our work on salmon could indicate amyloid plaques may form around dead neurons somewhat like Band-Aids, preventing toxins produced by dead neurons from reaching healthy neurons.''

Alzheimer's, the most common cause of dementia, affects an estimated 4 million Americans. Many neurologists believe that beta amyloid protein causes brain cells to degenerate and die.

Maldonado's team found that some areas of the salmon's brains did show the classic signs of neuron damage when the amyloid protein was also present. But other brain areas -- for instance, those used to help the fish find their way home to spawn -- functioned well despite the plaques, Maldonado said. The researchers studied salmon because the fish have remarkably similar aging symptoms, including brain decay, cardiovascular disease, muscle atrophy and skin lesions.

They castrated young kokanee salmon and found they lived to be 7 to 9 years old, instead of dying at age 2 or 3 as normal kokanee do. The researchers said that suggested salmon have an ''aging alarm'' timed to go off at reproduction. They found that levels of a stress hormone known as cortisol surge just before the onset of the rapid aging process and death.

``Cortisol surges may help these fish metabolize sugar and produce enough energy to locate their home streams and reproduce, but the surges also eventually may trigger brain aging and death,'' Maldonado said. Other experiments have shown that cortisol spikes occur in Alzheimer patients, and they are known to kill certain areas of neurons in the brain. ``If we find that stress hormones cause amyloid plaques to form in salmon brains, that would be quite a breakthrough,'' said biology professor David Norris, who also worked on the study.

Comment (webmaster): Recall that Gibbs et al had this same idea some time back; no prion disease was found. This might be need to be revisited with these AD positive salmon: "Normal isoform of amyloid protein (PrP) in brains of spawning salmon," Mol Psychiatry. 1997 Mar;2(2):146-7 Gibbs CJ Jr, Bolis CL.

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