Amplification techniques and diagnosis of prion diseases.

Protein misfolding cyclic amplification assay (PMCA) and real-time quaking-induced conversion (RT-QuIC) are two amplification techniques based on the ability of PrPsc to induce a conformational change in PrP allowing the detection of minute amounts of PrPsc in body fluids or tissues. PMCA and RT-QuIC have different ability to amplify PrPsc from sporadic, variant and genetic forms of Creutzfeldt-Jakob disease (CJD). PMCA readily amplifies PrPsc from variant CJD (vCJD) tissue while RT-QuIC easily amplifies PrPsc from sporadic CJD (sCJD) patient tissues. In terms of diagnosis, this implies the possibility of distinguishing vCJD from sCJD and explains the wider use of RT-QuIC given the respective frequencies of vCJD and sCJD. The sensitivity values of RT-QuIC for the diagnosis of sCJD are comparable or higher than those of the other tests (EEG, MRI, detection of 14-3-3 or tau proteins in cerebrospinal fluid) but with a specificity close to 100%. These new diagnostic methods could also be useful for the diagnosis of other neurodegenerative diseases.

Long-standing prion dementia manifesting as posterior cortical atrophy.

Prion diseases commonly manifest with the phenotype of subacute myoclonic encephalopathy. However, genetic forms of prion disease may have prolonged evolution mimicking neurodegenerative disease. We present the clinical and neuropathological features of a family with an early and long-standing dementia manifesting with posterior cortical atrophy and related to a 120 bp insertional mutation of the prion protein gene. Two cases exhibited mixed prion and Aß pathology. The differential diagnosis with Alzheimer disease is discussed.

Telomere length and mitochondrial DNA copy number in bipolar disorder: identification of a subgroup of young individuals with accelerated cellular aging.

The 10-15-years decrease in life expectancy observed in individuals with bipolar disorder (BD) has been linked to the concept of accelerated cellular aging. Telomere length (TL) and mitochondrial DNA copy number (mtDNAcn) have been proposed as markers of cellular aging and comparisons between individuals with BD and healthy controls (HC) sometimes led to conflicting results. Previous studies had moderate sample sizes and studies combining these two markers into a single analysis are scarce. Using quantitative polymerase chain reaction, we measured both TL and mtDNAcn in DNA (peripheral blood) in a sample of 130 individuals with BD and 78 HC. Regression analyses, receiver operating characteristic (ROC), and clustering analyses were performed. We observed significantly lower TL and mtDNAcn in individuals with BD as compared to HC (respective decrease of 26.5 and 35.8%). ROC analyses showed that TL and mtDNAcn highly discriminated groups (AUC = 0.904 for TL and AUC = 0.931 for mtDNAcn). In the whole population, clustering analyses identified a group of young individuals (age around 36 years), with accelerated cellular aging (both shorter TL and lower mtDNAcn), which consisted mostly of individuals with BD (85.5%). The subgroup of patients with young age but accelerated aging was not characterized by specific clinical variables related to the course of BD or childhood maltreatment. However, patients in this subgroup were more frequently treated with anticonvulsants. Further characterization of this subgroup is required to better understand the molecular mechanisms and the risk factors of accelerated cellular aging in BD.

Quantification of surviving cerebellar granule neurones and abnormal prion protein (PrPSc) deposition in sporadic Creutzfeldt-Jakob disease supports a pathogenic role for small PrPSc deposits common to the various molecular subtypes.

AIMS: Neuronal death is a major neuropathological hallmark in prion diseases. The association between the accumulation of the disease-related prion protein (PrP(Sc) ) and neuronal loss varies within the wide spectrum of prion diseases and their experimental models. In this study, we investigated the relationships between neuronal loss and PrP(Sc) deposition in the cerebellum from cases of the six subtypes of sporadic Creutzfeldt-Jakob disease (sCJD; n=100) that can be determined according to the M129V polymorphism of the human prion protein gene (PRNP) and PrP(Sc) molecular types. METHODS: The numerical density of neurones was estimated with a computer-assisted image analysis system and the accumulation of PrP(Sc) deposits was scored. RESULTS: The scores of PrP(Sc) immunoreactive deposits of the punctate type (synaptic type) were correlated with neurone counts - the higher the score the higher the neuronal loss - in all sCJD subtypes. Large 5- to 50-µm-wide deposits (focal type) were found in sCJD-MV2 and sCJD-VV2 subtypes, and occasionally in a few cases of the other studied groups. By contrast, the highest scores for 5- to 50-µm-wide deposits observed in sCJD-MV2 subtype were not associated with higher neuronal loss. In addition, these scores were inversely correlated with neuronal counts in the sCJD-VV2 subtype. CONCLUSIONS: These results support a putative pathogenic role for small PrP(Sc) deposits common to the various sCJD subtypes. Furthermore, the observation of a lower loss of neurones associated with PrP(Sc) type-2 large deposits is consistent with a possible 'protective' role of aggregated deposits in both sCJD-MV2 and sCJD-VV2 subtypes.

Signal transduction through prion protein.

The cellular prion protein PrPc is a glycosylphosphatidylinositol-anchored cell-surface protein whose biological function is unclear. We used the murine 1C11 neuronal differentiation model to search for PrPc-dependent signal transduction through antibody-mediated cross-linking. A caveolin-1-dependent coupling of PrPc to the tyrosine kinase Fyn was observed. Clathrin might also contribute to this coupling. The ability of the 1C11 cell line to trigger PrPc-dependent Fyn activation was restricted to its fully differentiated serotonergic or noradrenergic progenies. Moreover, the signaling activity of PrPc occurred mainly at neurites. Thus, PrPc may be a signal transduction protein.

Diagnosis associated with Tau higher than 1200 pg/mL: Insights from the clinical and laboratory practice.

CONTEXT: Cerebrospinal fluid (CSF) biomarkers are valuable tools for the diagnosis of neurological diseases. We aimed to investigate within a retrospective multicentric study the final diagnosis associated with very high CSF Tau levels and to identify patterns of biomarkers that would differentiate them in clinical practice, to help clinical biologists into physicians' counseling. PATIENTS AND METHODS: Within the national multicentric network ePLM, we included 1743 patients from January 1, 2008, to December 31, 2013, with CSF biomarkers assayed by the same Innotest assays (protein Tau, phospho-Tau [pTau], and Aß 1-42). We identified 205 patients with protein Tau concentration higher than 1200 pg/mL and final diagnosis. RESULTS: Among those patients, 105 (51.2%) were suffering from Alzheimer's disease, 37 (18%) from sporadic Creuztfeldt-Jakob disease, and 63 (30.7%) from other neurological diseases including paraneoplastic/ central nervous system tumor, frontotemporal dementia, other diagnoses, amyloid angiopathy, Lewy body dementia, and infections of the central nervous system. Phospho-Tau, Aß1-42 and Aß1-42/pTau values differed significantly between the three groups of patients (p < .001). An Aß1-42/pTau ratio between 4.7 and 9.7 was suggestive of other neurological diseases (threshold in AD: 8.3). CSF 14-3-3 was useful to discriminate Alzheimer's disease from Creuztfeldt-Jakob disease in case of Aß1-42 concentrations <550 pg/mL or pTau>60 pg/mL. CONCLUSION: This work emphasizes the interest of a well-thought-out interpretation of CSF biomarkers in neurological diseases, particularly in the case of high Tau protein concentrations in the CSF.

[Alzheimer's disease cerebro-spinal fluid biomarkers: A clinical research tool sometimes useful in daily clinical practice of memory clinics for the diagnosis of complex cases]. / Les biomarqueurs du liquide cérébro-spinal dans la maladie d'Alzheimer : un outil de recherche utile dans la pratique clinique courante des consultations mémoire pour les cas complexes.

The role of biomarkers in clinical research was recently highlighted in the new criteria for the diagnosis of Alzheimer's disease. Cerebro-spinal fluid (CSF) biomarkers (total Tau protein, threonine 181 phosphorylated Tau protein and amyloid Aß1-42 peptide) are associated with cerebral neuropathological lesions observed in Alzheimer's disease (neuronal death, neurofibrillary tangle with abnormal Tau deposits and amyloid plaque). Aß1-40 amyloid peptide dosage helps to interpret Aß1-42 results. As suggested in the latest international criteria and the French HAS (Haute Autorité de santé) recommendations, using theses CSF biomarkers should not be systematic but sometimes could be performed to improve confidence about the diagnostic of Alzheimer's disease in young subjects or in complex clinical situations. Future biomarkers actually in development will additionally help in diagnostic process (differential diagnosis) and in prognostic evaluation of neurodegenerative diseases.

Acute pain Factors predictive of post-operative pain and opioid requirement in multimodal analgesia following knee replacement.

BACKGROUND: Despite the development of multimodal analgesia for postoperative pain management, opioids are still required for effective pain relief after knee arthroplasty. We aimed to identify the determinants of post-operative pain intensity and post-operative opioid requirement in this context. METHODS: In this observational prospective study, we recorded patient characteristics, pre-operative pain intensity, anxiety and depression levels, sensitivity and pain thresholds in response to an electrical stimulus, and mu-opioid receptor (OPRM1) and catechol-O-methyltransferase (COMT) single-nucleotide polymorphisms. Multivariate linear regression models were used to identify predictors of post-operative pain at rest and opioid requirement. RESULTS: We included 109 patients. Pre-operative pain at rest (p = 0.047), anxiety level (p = 0.001) and neuropathic pain symptoms (p = 0.030) were independently and positively associated with mean post-operative pain intensity adjusted for mean post-operative morphine equivalent dose (MED). Mean post-operative pain intensity at rest was lower (p = 0.006) in patients receiving celecoxib and pregabalin in the post-operative period, with all other variables constant. Mean post-operative MED over 5 days was low, but highly variable (78.2 ± 32.1 mg, from 9.9 to 170 mg). Following adjustment for mean post-operative pain intensity, it was independently negatively correlated with age (p = 0.004), and positively correlated with associated paracetamol treatment (p = 0.031). No genetic effect was detected in our sample. CONCLUSIONS: Our findings suggest that clinicians could use the pre-operative pain profile, in terms of anxiety levels, neuropathic pain symptoms, and chronic pre-operative pain intensity, to improve the efficacy of pain management after knee surgery.

Identification of three novel mutations (E196K, V203I, E211Q) in the prion protein gene (PRNP) in inherited prion diseases with Creutzfeldt-Jakob disease phenotype.

Inherited prion diseases are characterized by mutations in the PRNP gene encoding the prion protein (PrP). As the other sporadic or infectious prion disease forms, they are almost all characterized by the accumulation in the brain of an abnormal misfolded form of the patient's PrP. Brain extracts can often transmit the disease once inoculated in a recipient animal. Inherited prion diseases with Creutzfeldt-Jakob disease (CJD) phenotype are autosomal forms, although sporadic cases have been reported. We report three novel mutations of the PRNP gene in unrelated patients with clinical and histopathologic features of CJD. The three mutations were missense: c635G>A (E196K), c656G>A (V203I) and c680G>C (E211Q). Familial history of neurologic disorders was evidenced for patients carrying the E196K and E211Q mutations. E196K would be predicted to have more severe effects on protein stability than V203I and E211Q. These mutations expand the spectrum of mutations in PRNP and reduce the proportion of CJD patients in whom genetic alterations have not been found.

Possible association between serotonin transporter gene polymorphism and violent suicidal behavior in mood disorders.

BACKGROUND: Genes involved in the serotonin system are major candidates in association studies of suicidal behavior. In this case-control study we investigated whether the serotonin transporter (5-HTT) gene encoding the protein responsible for the reuptake of serotonin from the synapse after its release from serotonergic neurons is a susceptibility factor for suicidal behavior. METHODS: A functional polymorphism of the 5-HTT gene (a 44-base pair insertion/deletion in the 5-HTT-linked polymorphic region [5-HTTLPR]) was studied in a population of 237 consecutive patients with affective disorder (unipolar or bipolar) and 187 control subjects. Ninety-nine patients had attempted suicide at least once, of whom 26 made a violent attempt. RESULTS: No association was found between the "s" allele of the 5-HTTLPR and suicide attempt; however, there was a significant difference in allele distributions between patients who had made violent suicide attempts and control subjects. CONCLUSIONS: A genetic variant of the 5-HTT gene may predispose individuals to violent suicidal behavior. The precise phenotype associated with the 5-HTT gene is unclear, and therefore further studies are required to replicate these findings.

Distribution of the M129V polymorphism of the prion protein gene in a Turkish population suggests a high risk for Creutzfeldt-Jakob disease.

A polymorphism (M129V) at codon 129 of the prion protein gene (PRNP) results in either a methionine residue (Met) or a valine residue (Val) and is known to determine susceptibility for the development of sporadic or acquired Creutzfeldt-Jakob disease (CJD). The distributions of M129V genotypes and alleles in various general populations have been reported and there are clear differences between Western Europeans and East Asians. We analysed the coding sequence of the PRNP gene in 100 healthy Turkish subjects to determine whether the distributions of the M129V genotypes and alleles or other PRNP gene variants in the Turkish population differ from those in other normal populations. Three known polymorphisms but no other gene variants were detected in the PRNP coding sequence of the Turkish individuals. Genotype frequencies at codon 129 were 57% Met/Met, 34% Met/Val and 9% Val/Val, with an allele frequency of 0.740:0.260 Met:Val. These distributions are considerably different from those reported for other normal populations residing in Western Europe and East Asia, except in Crete. The higher frequency of 129 Met-homozygotes in Turkey than in Western Europe suggests that the Turkish are at greater risk of developing CJD.

Prion-like protein Doppel expression is not modified in scrapie-infected cells and in the brains of patients with Creutzfeldt-Jakob disease.

Doppel protein has been discovered in prnp knock-out mouse lines, with overproduction of this protein in the brain causing ataxia and neurodegeneration. We investigated whether Doppel expression (i) affected or was affected by the course of prion propagation in neuroblastoma cells, or (ii) modulated Creutzfeldt-Jakob disease pathogenesis. No change in Doppel production was detected in N2a cells, before or after infection. Transient murine Doppel gene expression had no effect on N2a viability or PrP(Sc) production. A sensitive immunometric assay revealed low levels of Doppel in human brain, reflecting weak transcription of the corresponding gene. No difference in brain Doppel levels was observed between Creutzfeldt-Jakob disease patients and controls, adding further evidence that Doppel is unlikely to be involved in prion disease pathogenesis.

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.

Novel approaches in diagnosis and therapy of Creutzfeldt-Jakob disease.

The scrapie prion protein, PrP(Sc), as well as its peptide fragment, PrP106-126, are toxic on neuronal cells, resulting in cell death by an apoptotic, rather than necrotic mechanism. The apoptotic process of neuronal cells induced by prion protein supports diagnosis and offers potential targets for therapeutic intervention of the prion diseases. Among the cerebrospinal fluid (CSF) proteins, which may serve as markers of neuronal cell death associated with prion diseases, the 14-3-3 protein(s) turned out to be the most promising one. A new sensitive assay allows the detection of even small changes in the normally low levels of these proteins. In vitro, the toxic effects displayed by PrP(Sc) and its peptide fragment can be blocked by antagonists of N-methyl-D-aspartate (NMDA) receptor channels, like Memantine. Also Flupirtine, a non-opiod analgesic drug, which is already in clinical use, was found to display in vitro a strong cytoprotective effect on neurons treated with PrP(Sc) or PrP106-126. This drug acts like a NMDA receptor antagonists, but does not bind to the receptor. Clinical trials on prion diseases with Flupirtine are in progress. Flupirtine was found to enhance the intracellular levels of the antiapoptotic protein Bcl-2 and the antioxidative agent glutathione (GSH). Due to its favourable pharmacokinetic profile, Flupirtine is considered to be a promising drug to prevent neuronal death in Creutzfeldt-Jakob disease (CJD) and other neurodegenerative disorders occurring with age, e.g. Alzheimer's disease.

Demyelinating peripheral neuropathy with Creutzfeldt-Jakob disease and mutation at codon 200 of the prion protein gene.

We performed a study of the distribution of PrP27-30, the proteinase-K-resistant form of prion protein, in the central and peripheral nervous system of a patient with a Glu200Lys mutation of the prion protein gene, cerebellar ataxia, subcortical dementia, rigidity, and demyelinating peripheral neuropathy. In the CNS, there was neuron loss and spongy degeneration, principally in the cerebellum, and with progressively lower density in the caudate nucleus, thalamus, temporal cortex, frontal cortex, and brainstem. Evaluation of the expression of PrP27-30 by Western blot showed that its distribution correlated with the intensity of the lesions in these regions. In contrast, we did not detect PrP27-30 in the peripheral nervous system where lesions consisted of demyelination, and remyelination that predominated in the proximal nerve trunks and roots.

Diagnosis of Creutzfeldt-Jakob disease: effect of clinical criteria on incidence estimates.

OBJECTIVE: To assess the effect of usage of three different versions of Creutzfeldt-Jakob disease (CJD) diagnostic criteria on estimates of CJD incidence. METHODS: A total of 428 patients referred for suspected sporadic CJD between 1991 and 1997 were classified according to different criteria to be compared after analysis of medical records. Specificity, sensitivity, and positive and negative predictive values were calculated for each set of criteria in the subgroup of patients with a postmortem examination. Positive and negative predictive values of the clinical diagnosis were applied to cases without postmortem examination. Subsequently, the true number of cases of CJD among the referred cases was estimated. RESULTS: By comparison with the French and European study criteria, the Masters' criteria showed higher sensitivity but lower specificity and positive predictive value. Comparison with an estimate of the true total number of CJD cases showed that Masters' criteria overestimated the incidence by 7%, whereas the French and the European study criteria led to an underestimate of 12%. Detection of the 14-3-3 protein in CSF, considered as an additional diagnostic criterion for clinically probable CJD, resulted in a slight increase in the estimated incidence when the French or European study criteria were applied. CONCLUSIONS: Different diagnostic criteria could lead to an under- or overestimation of the true incidence of CJD. Therefore, comparisons of CJD incidence in different countries should rely on diagnostic classifications using identical criteria. Taking into account 14-3-3 protein detection as a criterion for probable CJD will result in a small increase in the estimated CJD incidence.

Molecular genetics of prion diseases in France. French Research Group on Epidemiology of Human Spongiform Encephalopathies.

Human prion diseases are characterized by the accumulation in the brain of an abnormal form of the prion protein. Prion protein polymorphisms seem to play a key role in the pathogenesis of these diseases, probably by enhancing the amyloidogenic properties of the protein. We performed prion protein gene (PRNP) coding sequence analysis in 57 French subjects with Creutzfeldt-Jakob disease (CJD) and found a mutation of the PRNP coding sequence in nine subjects (15.8%); the mutation corresponded with a known family history of CJD in only three of these subjects. In 41 definite and probable cases without known PRNP mutations, codon 129 genotyping revealed an excess of the homozygous 129Met/Met genotype corresponding to a 3.4-fold increased risk of developing CJD when compared with the two other genotypes. We also found that the 129Val/Val genotype, which mainly governs susceptibility to iatrogenic CJD, does not seem to predispose to sporadic CJD.

A new point mutation in the prion protein gene at codon 210 in Creutzfeldt-Jakob disease.

We screened 16 cases of sporadic Creutzfeldt-Jakob disease (CJD) and 28 healthy control subjects to detect possible polymorphisms in their prion protein gene (PRNP). The molecular analysis of the PRNP coding sequence was performed using denaturing gradient gel electrophoresis of polymerase chain reaction products and direct sequencing. We identified (1) a silent mutation at codon 177 in a healthy individual, (2) a codon 200 glutamate-to-lysine substitution in a 48-year-old CJD-affected Libyan Jew, and (3) a G-to-A point substitution at codon 210, leading a valine-to-isoleucine change, in a 63-year-old French CJD patient. This new mutation occurs in a highly conserved part of the PRNP coding sequence, close to the known CJD-associated codon 200 mutation, and might be linked to a symptomatologic and neuropathologic pattern of typical sporadic CJD. This mutation was also present in a sister of the patient who died at the age of 67 without neurologic symptomatology.