Biomedical subjects

Ruth Gabizon

Publications and source records attributed to Ruth Gabizon.

13 recordsLinked to original sources

PubMed2006

Prion urine comprises a glycosaminoglycan-light chain IgG complex that can be stained by Congo red.

Light chain IgG, a known amyloidotic protein, is present in the urine of prion disease affected individuals in a protease resistant form. In addition, it was shown recently that prion urine samples comprise a significant excess of glycosaminoglycans. Since amyloidotic proteins and glycosaminoglycans are the major components of amyloid aggregates, a Congo red dot blot assay was developed for detection of Creutzfeldt-Jacob disease (CJD) in urine. This assay was also positive for about 10% of patients suffering from diseases such as Alzheimer disease, cerebrovascular attacks and multiple sclerosis, but negative for healthy controls. Both glycosaminoglycans and proteins such as light chain IgG were required for the binding of Congo red to the urine fractions, as shown by the fact that Proteinase K digestion of the samples either after guanidine or after choindrotinase abolished the Congo red signal from the CJD samples.

Carrier Proteins↗

PubMed2006

Urine from scrapie-infected hamsters comprises low levels of prion infectivity.

The question of whether prion diseases can be transmitted by body fluids has important epidemiological, environmental and economical implications. In this work, we set to investigate whether urine collected from scrapie-infected hamsters can transmit fatal or subclinical infectivity to normal hamsters. After prolonged incubation times ranging from 300 to 700 days, a small number of animals inoculated with scrapie urine succumbed to scrapie disease, and several asymptomatic hamsters presented low levels of PrP(Sc) in their brains. In addition, most of the asymptomatic hamsters inoculated with scrapie urine, as opposed to those inoculated with normal urine, presented extensive gliosis as well as protease-resistant light chain IgG in their urine, a molecule shown by us and others to be a surrogate marker for prion infection. Our results suggest that urine from scrapie-infected hamsters can transmit a widespread subclinical disease that in some cases develops into fatal scrapie.

Animals↗

PubMed2005

The metabolism of glycosaminoglycans is impaired in prion diseases.

It is well established that the conversion of PrP(C) to PrP(Sc) is the key event in prion disease biology. In addition, several lines of evidence suggest that glycosaminoglycans (GAGs) and in particular heparan sulfate (HS) may play a role in the PrP(C) to PrP(Sc) conversion process. It has been proposed that PrP(Sc) accumulation in prion diseases may induce aberrant activation of lysosomal activity, which has been shown to result in neurodegeneration in a number of diseases, especially lysosomal storage disorders. Among such diseases, only the ones resulting from defects in GAGs degradation are accompanied by secretion of large amounts of GAG metabolites in urine. In this work, we show that GAGs are secreted in the urine of prion-infected animals and humans, and surprisingly, also in the urine of mice ablated for the PrP gene. We hypothesize that both the presence of PrP(Sc) or the absence of PrP(C) may alter the metabolism of GAGs.

Animals↗

PubMed2005

PrPSc incorporation to cells requires endogenous glycosaminoglycan expression.

Many lines of evidence suggest an interaction between glycosaminoglycans (GAGs) and the PrP proteins as well as a possible role for GAGs in prion disease pathogenesis. In this work, we sought to determine whether the PrP-GAG interaction affects the incorporation of PrP(Sc) (the scrapie isoform of PrP) to normal cells. This may be the first step in prion disease pathogenesis. To this effect, we incubated proteinase K-digested hamster scrapie brain homogenates with several lines of Chinese hamster ovary (CHO) cells in the presence or absence of heparin. Our results show that over a large range of PrP(Sc) concentrations the binding of PrP(Sc) to wild type CHO cells, which do not express detectable PrP, was equivalent to the binding of PrP(Sc) to CHO cells overexpressing PrP. A significant part of PrP(Sc) binding to both lines could be inhibited by heparin. Additional evidence that PrP(Sc) binding to cells was dependent on the presence of GAGs could be concluded from the fact that the binding of PrP(Sc) to CHO cells missing GAGs on the cell surface was significantly reduced. Interestingly, preincubation of scrapie brain homogenate with heparin before intraperitoneal inoculation into normal hamsters resulted in a significant delay in prion disease manifestation.

Animals↗

PubMed2005

Characterization of light chain immunoglobulin in urine from animals and humans infected with prion diseases.

The necessity of a non-invasive in-vivo test for prion diseases has become more apparent since the transmission of vCJD from the blood of a healthy individual incubating the disease. Here we show that prion urine comprises an array of protease resistant peptides, among them light chain immunoglobulin (LC). This was observed by sequencing gel bands comprising hamster urine samples, as well as by immunoblotting of similar samples with anti mouse IgG reagents for hamster samples, or with anti human IgG reagents for human samples. Our result suggests that urine samples from CJD patients can be identified by the presence of protease resistant proteins such as LC.

Amino Acid Sequence↗

PubMed2005

Inhibition of P53-related apoptosis had no effect on PrP(Sc) accumulation and prion disease incubation time.

Results from several laboratories indicate that apoptosis via the P53 pathway is involved in prion disease pathogenesis. Prion diseases, among them scrapie and BSE, are a group of fatal neurodegenerative disorders associated with the conversion of PrP(C) to PrP(Sc), its conformational abnormal isoform. In this work, we tested whether an established anti-apoptotic reagent, PFT, which has been shown in different systems to inhibit P53 activity, can delay the outbreak of prion disease in infected animals. Our findings indicate that although PFT efficiently reduced caspase 3 expression in brains from scrapie sick hamsters, as well as inhibited PrP(Sc) accumulation in cell culture, it had no effect on disease incubation time or PrP(Sc) accumulation in vivo. We conclude that the P53 dependent apoptosis may not be an obligatory mechanism for prion disease-induced cell death.

Animals↗

PubMed2003

Novel heparan mimetics potently inhibit the scrapie prion protein and its endocytosis.

During prion diseases the normal prion protein PrP(C) is refolded into an abnormal conformer PrP(Sc). We have studied the PrP(Sc) inhibiting activity of a library of synthetic heparan mimetic (HM) biopolymers. HMs are chemically derived dextrans obtained by successive substitutions with carboxymethyl, benzylamide, and sulfate groups on glucose residues. Some HMs eliminated PrP(Sc) from prion-infected cells after a 5 day course at 100 ng/ml and were 15 x potent than pentosan sulfate in this system. The anti-PrP(Sc) activity of HMs correlated with the degree of sulfation but was increased by benzylamidation. HMs did not reduce the synthesis of PrP(C) nor its attachment to lipid rafts, but instead blocked its conversion into PrP(Sc). The anti-PrP(Sc) HMs also prevented the uptake of prion rods by cultured cells. HMs may thus block the interaction of PrP(Sc) with a putative cellular receptor, possibly heparan sulfate. HMs provide an attractive chemical approach for the synthesis of TSE therapeutic and prophylactic reagents.

Animals↗

PubMed2003

Copper binding to PrPC may inhibit prion disease propagation.

Although it has been well established that PrP(C), the normal isoform of PrP(Sc), is a copper-binding protein, the role of this metal in the function of PrP(C) as well as in prion disease pathology remains unclear. Here, we show that when scrapie-infected neuroblastoma cells were cultured in the presence of copper, the accumulation of PrP(Sc) in these cells was markedly reduced. In addition, our results indicate that when normal neuroblastoma cells were cultured in the presence of copper ions, they could no longer bind and internalize PrP(Sc). In another set of experiments, copper was added to the drinking water of normal and scrapie-infected hamsters. Our results show that administration of copper to normal hamsters induced cerebellar PrP(C) accumulation. Most important, a significant delay in prion disease onset was observed when scrapie-infected hamsters were treated with copper. As shown before for neuroblastoma cells, also in vivo most of the copper-induced accumulation of PrP(C) was intracellular. We hypothesized that PrP(C) internalization by copper may hinder PrP(Sc) interaction with this molecule, and thereby affect prion disease propagation.

Animals↗

PubMed2003

Dimethyl sulfoxide delays PrP sc accumulation and disease symptoms in prion-infected hamsters.

PrP(Sc), an aberrantly folded protein, is the only identified component of the prion, an agent causing fatal neurodegenerative diseases such as scrapie and bovine spongiform encephalopathy. Dimethyl sulfoxide (DMSO) has been shown to reduce the accumulation of PrP(Sc) in scrapie-infected (ScN2a) cells, and to inhibit its aggregation in vitro. In humans, DMSO was used successfully in the treatment of various peripheral amyloidotic diseases. Here we show that administration of DMSO to scrapie-infected hamsters significantly prolonged disease incubation time, as well as delayed the accumulation of PrP(Sc) in Syrian hamster brains. Interestingly, administration of DMSO to scrapie sick hamsters resulted in increased clearance of protease-resistant PrP in their urine. We conclude that although DMSO by itself may not be sufficient to cure prion diseases, it may be considered as a component in a 'cocktail' drug approach for these disorders. Also, urine PrP testing should be considered for the assessment of treatment efficacy.

Animals↗

PubMed2002

Doppel and PrP(C) do not share the same membrane microenvironment.

Doppel is a paralog of the normal prion protein, PrP(C). It has been suggested that Doppel can compensate for the absence of PrP(C) in PrP(0/0) mice. In this work, we tested whether Doppel and PrP(C) share the same cell location, thereby sharing the same neighboring cell components, probably required to share the same cell function. Our results show that, at detergent conditions in which membrane rafts were intact, neither PrP(C) and Doppel co-immunoprecipitate with the appropriate antibodies, nor was Doppel retained by a Cu(2+)IMAC resin, as PrP(C) does. This indicates that, although Doppel is a raft-associated protein as is PrP(C), both proteins are not present in the same membrane microenvironment, and they probably do not perform the same function.

Animals↗

PubMed2002

Protease-sensitive scrapie prion protein in aggregates of heterogeneous sizes.

The pathological prion protein PrP(Sc) is the only known component of the infectious prion. In cells infected with prions, PrP(Sc) is formed posttranslationally by the refolding of the benign cell surface glycoprotein PrP(C) into an aberrant conformation. The two PrP isoforms possess very different properties, as PrP(Sc) has a protease-resistant core, forms very large amyloidic aggregates in detergents, and is only weakly immunoreactive in its native form. We now show that prion-infected rodent brains and cultured cells contain previously unrecognized protease-sensitive PrP(Sc) varieties. In both ionic (Sarkosyl) and nonionic (n-octyl beta-D-glucopyranoside) detergents, the novel protease-sensitive PrP(Sc) species formed aggregates as small as 600 kDa, as measured by gel filtration. The denaturation dependence of PrP(Sc) immunoreactivity correlated with the size of the aggregate. The small PrP(Sc) aggregates described here are consistent with the previous demonstration of scrapie infectivity in brain fractions with a sedimentation coefficient as small as 40 S [Prusiner et al. (1980) J. Neurochem. 35, 574-582]. Our results demonstrate for the first time that prion-infected tissues contain protease-sensitive PrP(Sc) molecules that form low MW aggregates. Whether these new PrP(Sc) species play a role in the biogenesis or the pathogenesis of prions remains to be established.

Animals↗

PubMed2002

Valproic acid treatment results in increased accumulation of prion proteins.

PrP(Sc), the only identified component of the prion, is an aberrant isoform of PrP(C), a glycoprotein of unknown function. In this study, it was shown that valproic acid, a widely used antiepileptic drug, can cause an increase of several orders of magnitude in the accumulation of PrP(C) in normal neuroblastoma cells (N2a), and of both PrP isoforms in scrapie infected neuroblastoma cells (ScN2a). Although preliminary results indicate that valproic acid administration to hamsters inoculated with prions had no significant effect on disease incubation time, it is suggested that administration of valproic acid to humans at risk of developing Creutzfeldt-Jakob disease should be evaluated with caution.

Animals↗

PubMed2002

The binding of prion proteins to serum components is affected by detergent extraction conditions.

As many GPI anchored proteins, PrP(C) and its abnormal conformer PrP(Sc), are inserted into membrane microdomains known as rafts. Upon raft disruption, PrP(C) becomes soluble, while PrP(Sc) aggregates into insoluble structures. It was recently published that, as opposed to PrP(C), PrP(Sc), as well as its protease resistant core PrP27-30, can bind specifically to plasminogen and other serum components. These findings were suggested to have important physiological implications in transmissible spongiform encephalopathies (TSE) diagnosis and pathogenesis. In this work, we show that the binding of PrP(Sc) or PrP 27-30 to serum proteins occurs only at specific detergent combinations, in which disease associated PrPs are present in aggregated structures. At detergent conditions in which rafts are intact, it is actually PrP(C.) that binds to blood proteins, albeit not directly, but through neighboring rafts components. Our results therefore indicate that the binding of PrP(Sc) to blood components has no physiological relevance.

Animals↗