Jean-Louis Laplanche

Distinct molecular phenotypes in bovine prion diseases

Bovine spongiform encephalopathy (BSE) in cattle, the most likely cause of variant Creutzfeldt–Jakob disease in humans, is thought to be caused by a unique infectious agent, with stable features, even when transmitted to other species. Here, we show the existence of an atypical molecular phenotype among cattle diagnosed with BSE in France. Following western blot analysis, three cases showed unusual features of the electrophoretic profiles of the protease‐resistant prion protein (PrPres) accumulating in the brain. The PrPres patterns were similar in these three atypical cases, showing a higher molecular mass of unglycosylated PrPres and strong labelling by P4 monoclonal antibody compared to 55 typical BSE cases. This finding suggests either some phenotypic modifications of PrPres following infection by the BSE agent or the existence of alternative origins of such diseases in cattle.

V180I mutation of the prion protein gene associated with atypical PrPSc glycosylation

A valine to isoleucine mutation at residue 180 was identified in a French patient with Creutzfeldt-Jakob disease (CJD). The mutation is located in the close vicinity of one of the two N-glycosylation sites of the cellular prion protein (PrP(C)). Western blot analysis revealed accumulation in the brain of the pathogenic proteinase K-resistant PrP (PrP(Sc)) isoform with the notable absence of the diglycosylated band. The mutant protein expressed in CHO cells was correctly glycosylated, suggesting that the atypical glycosylation pattern of PrP(Sc) was not due to the mutation at position 180. These results suggest that the diglycosylated form of the mutant PrP(180I) prevents its conversion into the pathogenic mutant form PrP(Sc180I), supporting a central role of N-linked glycan chains in the PrP conversion process.

Cerebrospinal Fluid PKR Level Predicts Cognitive Decline in Alzheimer’s Disease

The cerebrospinal fluid (CSF) levels of the proapoptotic kinase R (PKR) and its phosphorylated PKR (pPKR) are increased in Alzheimer’s disease (AD), but whether CSF PKR concentrations are associated with cognitive decline in AD patients remain unknown. In this study, 41 consecutive patients with AD and 11 patients with amnestic mild cognitive impairment (aMCI) from our Memory Clinic were included. A lumbar puncture was performed during the following month of the clinical diagnosis and Mini-Mental State Examination (MMSE) evaluations were repeated every 6 months during a mean follow-up of 2 years. In AD patients, linear mixed models adjusted for age and sex were used to assess the cross-sectional and longitudinal associations between MMSE scores and baseline CSF levels of Aβ peptide (Aβ 1-42), Tau, phosphorylated Tau (p-Tau 181), PKR and pPKR. The mean (SD) MMSE at baseline was 20.5 (6.1) and MMSE scores declined over the follow-up (-0.12 point/month, standard error [SE]u200a=u200a0.03). A lower MMSE at baseline was associated with lower levels of CSF Aβ 1–42 and p-Tau 181/Tau ratio. pPKR level was associated with longitudinal MMSE changes over the follow-up, higher pPKR levels being related with an exacerbated cognitive deterioration. Other CSF biomarkers were not associated with MMSE changes over time. In aMCI patients, mean CSF biomarker levels were not different in patients who converted to AD from those who did not convert.These results suggest that at the time of AD diagnosis, a higher level of CSF pPKR can predict a faster rate of cognitive decline.

Prion protein and neuronal differentiation: quantitative analysis of prnp gene expression in a murine inducible neuroectodermal progenitor

The biological function of the cellular prion protein, PrP(c), is currently unknown. The presence of PrP(c) transcripts in the developing neural tube from embryonic day 13.5 and the predominant expression of PrP(c) in the adult brain is suggestive of a role in the onset and/or modulation of neuronal functions. We took advantage of the bipotential neuroectodermal 1C11 cell line to monitor PrP(c) expression during its bioaminergic differentiations. The F9-derived 1C11 precursor cell line displays a stable and immature phenotype in the absence of extracellular signal and, upon induction, has the capacity to acquire a complete serotonergic or noradrenergic phenotype, the two pathways being mutually exclusive. A real-time quantitative PCR assay was developed to assess PrP(c) gene expression at definite times of the two programs that correspond to sequential acquisition of neurotransmitter-specific functions. 1C11 cells and their differentiated progenies express significant amounts of PrP transcripts and of the corresponding protein. A unique decrease in prnp gene expression is observed upon entry into the serotonergic pathway, correlating with a downregulation at the protein level. Moreover, nerve growth factor (NGF) is shown to induce a decrease in the level of prnp gene expression along the serotonergic - but not the noradrenergic - pathway. Our study accurately establishes that prnp gene expression (i) is strongly upregulated concomitantly with cell fate restriction of multipotential cells towards the neural lineage; (ii) is differentially regulated along the serotonergic versus noradrenergic differentiation program of a unique neuroectodermal progenitor. The 1C11 cell line may provide a new tool for studying prion infectivity in a well-defined neuronal context.

Increased Cerebrospinal Fluid Levels of Double-Stranded RNA-Dependant Protein Kinase in Alzheimer's Disease

BACKGROUNDnThe pathological hallmarks of Alzheimers disease (AD) include accumulation of amyloid-β (Aß) peptide forming extracellular senile plaques, neurofibrillary tangles made of hyperphosphorylated tau protein with neuronal loss. Aβ peptide (1-42), total tau (T-tau), and phosphorylated tau at threonine 181 (p181tau) levels in the cerebrospinal fluid (CSF) are now validated biomarkers. The proapoptotic kinase R (PKR), is activated by Aβ accumulates in degenerating neurons in AD brains and controls protein synthesis and indirectly tau phosphorylation.nnnMETHODSnIn a prospective cohort study, the CSF of 91 patients were studied (AD: 45; amnestic mild cognitive impairment: 11; neurological disease control subjects [NDC]: 35). The levels of total PKR (T-PKR), phosphorylated PKR (pPKR), Aß 1-42, T-tau, and p181tau were assessed by immunoblotting or enzyme-linked immunosorbent assay methods. Receivers operating characteristic curves were used to examine the discriminatory power of T-PKR, pPKR, and pPKR/T-PKR ratio between AD and NDC patients.nnnRESULTSnTotal PKR and pPKR concentrations were elevated in AD and amnestic mild cognitive impairment subjects. We have determined a pPKR value (optical density units) that could discriminate AD patients from control subjects with a sensitivity of 91.1% and a specificity of 94.3%. Among AD patients, T-PKR and pPKR levels correlate with CSF p181tau levels. Some AD patients with normal CSF Aß, T-tau, or p181tau levels had abnormal T-PKR and pPKR levels.nnnCONCLUSIONSnThe evaluation of CSF T-PKR and pPKR can discriminate between AD patients and NDC and could help to improve the biochemical diagnosis of AD.

Mutation at codon 210 (V210I) of the prion protein gene in a North African patient with Creutzfeldt-Jakob disease

A point mutation at codon 210 of the prion protein gene (PRNP), resulting in the substitution of isoleucine for valine (V210I) has been found in a 54-year-old Moroccan patient affected with Creutzfeldt-Jakob disease (CJD). This patient is the first carrier of the PRNP V210I mutation reported from North Africa. The clinical presentation of the patient was rather similar to that seen in classical CJD, except that unusual early sensory symptoms were observed. The mother of the proband, aged 72, is a further example of an asymptomatic elderly carrier of the PRNP V210I mutation, suggesting an incomplete penetrance of the disease.

Genetic heterogeneity versus molecular analysis of prion susceptibility in neuroblasma N2a sublines

The neuroblastoma-derived cell line N2a is permissive to certain prion strains but resistant sublines unable to accumulate the pathological proteinase-K resistant form of the prion protein can be isolated. We compared for gene expression and phenotypes different N2a sublines that were susceptible or resistant to the 22L prion strain. Karyotypes and comparative genomic hybridization arrays revealed chromosomal imbalances but did not demonstrate a characteristic profile of genomic alterations linked to prion susceptibility. Likewise, we showed that this phenotype was not dependent on the binding of PrPres, the expression of the prion protein gene, or on its primary sequence. We completed this analysis by looking using real-time quantitative PCR at the expression of a set of genes encoding proteins linked to prion biology. None of the candidates could account by itself for the infection phenotype, nevertheless sublines had distinct transcriptional profiles. Taken together, our results do not support a role for specific genomic abnormalities and possible candidate proteins in N2a prion susceptibility. They also reveal genetic heterogeneity among the sublines and serve as a guidance for further investigation into the molecular mechanisms of prion infection.

E - 17 Polymorphisme 129 dans une population de patients initialement suspects de MCJ

Introduction La maladie de Creuzfeldt-Jakob (MCJ) existe sous une forme sporadique, genetique ou acquise. Le codon 129 du gene de la proteine prion (PRNP) presente un polymorphisme biallelique codant soit une valine soit une methionine. Objectifs Etudier la repartition du polymorphisme 129, implique dans la susceptibilite a la MCJ, dans une population francaise de patients exclus a posteriori du diagnostic de maladie a prions (analyse retrospective ; 2000-2005). Methodes Le genotype au codon 129 a ete determine par PCR-RFLP ou DGGE chez 489 patients. Ont ete exclus les patients « perdus de vue » par le reseau pour lesquels un diagnostic n’a pu etre pose. Resultats . La frequence allelique de l’allele methionine est evaluee en France dans cette population a 0,641. Les patients heterozygotes representent 50,1 % des individus, les Met/Met 39,1 %, les Val/Val 10,8 %. Les homozygotes representent 49,9 % des patients. Discussion Chez les patients presentant une MCJ sporadique, les homozygotes representent pres de 80 % avec une predominance de genotype 129 Met/Met (Palmer, 1991). Tous les patients atteints de v-MCJ decris a ce jour sont homozygotes Met/Met au codon 129. La frequence que nous observons dans notre population est similaire a celle determinee dans les differentes populations europeennes de donneurs sains ainsi qu’aux donnees precedemment publiees en France. Conclusion La repartition du genotype au codon 129 de PRNP ne semble donc pas modifiee chez ces patients. Le genotype au codon 129 ne semble donc pas jouer un role majeur dans la susceptibilite a des demences autres que la MCJ.

E - 4 Sensibilité de la détection la protéine 14-3-3 pour le diagnostic de la maladie de Creutzfeldt-Jakob sporadique : données de 1997 à 2006

Introduction La MCJ sporadique represente 80 % des maladies a prions. Le diagnostic de certitude est fonde sur l’examen post mortem. La detection de la proteine 14-3-3, dans le LCR, est importante pour orienter le diagnostic. Objectifs Determiner la sensibilite de la detection de la proteine 14-3-3 dans les cas de MCJ sporadiques certaines. Evaluer l’interet d’un deuxieme prelevement de LCR en cas de premier prelevement negatif. Methodes Les patients atteints de MCJ sporadiques certaines, recenses par le reseau d’Epidemio-surveillance durant la periode 1997-2006, ont ete analyses. Seuls les patients ayant eu au moins une detection de la proteine 14-3-3 dans le LCR, par une technique de Western-blot manuelle qualitative, dans le laboratoire associe au CNR ont ete inclus. Resultats Parmi 453 cas de MCJ sporadiques certaines, 362 patients (80 %) avaient une detection de la proteine 14-3-3 positive (ou positive faible) des le premier prelevement. Parmi les 91 patients ayant un premier prelevement negatif (ou douteux), seuls 39 ont beneficie d’un second prelevement qui est devenu positif (ou positif faible) dans 18 cas (46 %). La sensibilite globale de la detection de la proteine 14-3-3 pouvait etre evaluee a 84 % dans ce groupe de MCJ sporadique certaine. Discussion La detection de la proteine 14-3-3 est un examen sensible. En cas de premiere detection negative, une seconde recherche, si les signes cliniques sont evocateurs de MCJ, semble souhaitable puisque la detection devient positive dans pres de 50 % des cas. La sensibilite etant inferieure a 100 %, en aucun cas une detection negative de la proteine 14-3-3 dans le LCR est un argument suffisant pour eliminer le diagnostic de MCJ sporadique. Conclusion La detection de la proteine 14-3-3 garde une bonne valeur pour le diagnostic de MCJ sporadique. Les auteurs remercient les cliniciens et neuropathologistes qui ont fourni les informations et les echantillons necessaires a cette etude.