Michele Halimi

Detection of 14-3-3 protein in the CSF of genetic Creutzfeldt-Jakob disease

The 14-3-3 protein, a protein involved in signal transduction, is present in the CSF of patients with Creutzfeldt-Jakob disease (CJD) and not in patients with other dementing diseases. We show here that this is also true for patients with E200K CJD, but not for healthy carriers of the mutation.

Copper Binding to the PrP Isoforms: a Putative Marker of Their Conformation and Function

ABSTRACT We show here that PrPC, the normal isoform of the prion protein (PrPSc), could be retained by a Cu2+-loaded resin through two different binding sites. Contrarily, PrPSc was not retained at all by such resin. This constitutes a new prion-specific property of PrPSc, which in addition to protease resistance and β-sheet content, may result from its aberrant conformation.

Presence of prion protein in peripheral tissues of Libyan Jews with Creutzfeldt‐Jakob disease

The prion protein (PrP) gene on chromosome 20 encodes a protein designated PrPC. An abnormal, protease-resistant isoform of PrPC, denoted PrPCJD or PrPSc, is present in the brains of patients with Creutzfeldt-Jakob disease (CJD). In Libyan Jews, CJD segregates with a point mutation at codon 200 of the PrP gene, resulting in the substitution of lysine for glutamate. In the present study, we examined the presence of PrP in fibroblasts and leukocytes derived from eight CJD patients with the codon 200 mutation. In cultured fibroblasts as well as in leukocytes, there was a significant increase in PrP as judged by immunocytochemistry in addition to immunoblotting. Most of the PrP in fibroblasts and leukocytes could be released from the external surface by phosphatidylinositol-specific phospholipase C, a property characteristic of PrPC. In leukocytes only, part of the protein was protease resistant, resembling PrPCJD. The concentration of PrP mRNA was similar in fibroblast lines derived from controls and CJD patients. These results suggest that in CJD patients carrying a mutation at codon 200 of the PrP gene, the metabolism of PrP, rather than PrP synthesis, is abnormal.

Effect of scrapie infection on the activity of neuronal nitric-oxide synthase in brain and neuroblastoma cells.

Nitric-oxide synthase (NOS) is responsible for the synthesis of nitric oxide which serves as a neural messenger in the central nervous system. NOS activity was markedly inhibited in brains of mice and hamsters and neuroblastoma cells infected with scrapie (ScN2a). The decrease in activity was in accordance with decreased NADPH-diaphorase-positive cells and decreased staining of NOS-positive cells demonstrated by specific anti-NOS antibodies. However, the specific nNOS mRNA in ScN2a was elevated when compared with normal neuroblastoma cells (N2a). Immunoblotting of fractions from these cell lines with an anti-nNOS monoclonal antibody revealed a band of nNOS from N2a and two bands with a lower molecular weight in ScN2a cells. Furthermore, NOS in ScN2a cells was insoluble in nondenaturing detergents. This insolubility is one of the landmark properties of PrPSc. It is, therefore, possible that nNOS in scrapie-infected cells and brains is aberrantly folded, resulting in an insoluble and inactive enzyme.

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.

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.

Differential allelic expression of PrP mRNA in carriers of the E200K mutation.

Creutzfeldt-Jakob disease (CJD) linked to the E200K mutation of the protein (PrP) gene presents with a wide range of age at disease onset. Since most patients are heterozygous for the mutation, we tested whether differential expression of mutant versus wild-type (wt) PrP may affect the age at disease onset in carriers of the mutation. We measured wt and mutant PrP protein and mRNA in Epstein-Barr virus (EBV)-transformed B cells of either E200K CJD patients or healthy E200K carriers. Our results suggest that while in most healthy carriers the expression of wt PrP was higher than that of E200K PrP, most of the E200K CJD patients express equal levels of both PrP proteins. Similar results were obtained for either PrP protein or PrP mRNA. These results suggest that preferential expression of PrP from the wt allele may modulate the outbreak of the disease in carriers of prion mutations. This notion is consistent with the results obtained in transgenic mice carrying a human PrP gene, which suggest that endogenous PrP protects mice from contracting scrapie after inoculation with human CJD brain. Similar mechanisms may prevail in other inherited diseases with variable phenotypes.

Properties of the Prion Proteins in Creutzfeldt Jakob Disease Patients Heterozygous for the E200K Mutation

Three groups of inherited prion diseases are recognized based on the clinical manifestations of these diseases: familial CJD Gerstmann-Straussler-Scheinker (GSS) disease and fatal familial insomnia (FFI)1. In Libyan Jews suffering from CJD, a point mutation which results in the substitution of K for E at residue 200 of PrP was identified2,3. This mutation was found in all definitely affected individuals and yields a maximum lod score of 4.854 The same mutation is also present in other clusters of the disease5.