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The physiological function of the cellular prion protein (PrP(C)) is still under intense investigation. It has been suggested that PrP(C) has a protective role in neuronal cells, particularly against environmental stress caused by reactive oxygen species (ROS). Here we analysed the acute effect of a major ROS, hydrogen peroxide (H(2)O(2)), on intracellular calcium homeostasis in cultured cerebellar granule cells and immortalized hippocampal neuronal cells. Both neuronal cell culture models showed that the rise in intracellular calcium following application of H(2)O(2) was strongly dependent on the presence of PrP(C). Moreover, the N-terminal octapeptide repeats of PrP(C) were required for this effect, because neuronal cells expressing a PrP(C) lacking the N-terminus resembled the PrP(C)-deficient phenotype. Neurones deficient of fyn kinase, or pharmacological inhibition of fyn, also abrogated the calcium response to H(2)O(2) treatment, indicating that fyn activation is a critical step within the PrP(C) signalling cascade. Finally, we identified a possible role of this PrP(C) signalling pathway in the neuroprotective response of PrP(C) to oxidative stress. In conclusion, we put forward the hypothesis that PrP(C) functions as a sensor for H(2)O(2), thereby activating a protective signalling cascade involving fyn kinase that leads to calcium release from intracellular stores.

Background: The measurement of biomarkers in cerebrospinal fluid (CSF) has gained increasing acceptance in establishing the diagnosis of some neurodegenerative diseases. Heart-type fatty acid-binding protein (H-FABP) was recently discovered in CSF and serum of patients with neurodegenerative diseases. Objective: We investigated H-FABP in CSF and serum alone and in combination with CSF tau protein to evaluate these as potential biomarkers for the differentiation between dementia with Lewy bodies (DLB) and Alzheimer’s disease (AD). Methods: We established H-FABP and tau protein values in a set of 144 persons with DLB (n = 33), Parkinson disease with dementia (PDD; n = 25), AD (n = 35) and nondemented neurological controls (NNC; n = 51). Additionally, serum H-FABP levels were analyzed in idiopathic Parkinson disease patients without evidence of cognitive decline (n = 45) using commercially available enzyme-linked immunosorbent assays. We calculated absolute values of H-FABP and tau protein in CSF and serum and established relative ratios between the two to obtain the best possible match for the clinical working diagnosis. Results: Serum H-FABP levels were elevated in DLB and PDD patients compared with NNC and AD subjects. To better discriminate between DLB and AD, we calculated the ratio of serum H-FABP to CSF tau protein levels. At the arbitrary chosen cutoff ratio ≧8 this quotient reached a sensitivity of 91% and a specificity of 66%. Conclusion: Our results suggest that the measurement of CSF tau protein, together with H-FABP quantification in serum and CSF, and the ratio of serum H-FABP to CSF tau protein represent marker candidates for the differentiation between AD and DLB.

Molecular typing in Creutzfeldt-Jakob disease (CJD) relies on the detection of distinct protease-resistant prion protein (PrP(Sc)) core fragments, which differ in molecular mass or glycoform ratio. However, the definition and correct identification of CJD cases with a co-occurrence of PrP(Sc) types remains a challenge. With antibodies recognizing a linear epitope in the octapeptide repeat PrP region, supposed to distinguish between the two major PrP(Sc) isoforms (ie, types 1 and 2), it was recently shown that all type 2 cases display an associated band with a type 1 migration pattern, which led to the conclusion that multiple PrP(Sc) types regularly coexist in CJD. We studied brain samples from 53 sporadic CJD and 4 variant CJD subjects using a high-resolution electrophoresis, a wide range of proteinase K (PK) activities, the 'type 1-selective' antibody 12B2, and several unselective antibodies. We found that the type 1-like band detected by 12B2 in all CJD subtypes associated with PrP(Sc) type 2 is not a PK-resistant PrP(Sc) core but rather matches the physicochemical properties of partially cleaved fragments, which result from the several PK cleavage sites included in the N-terminal portion of PrP(Sc). Furthermore, using gels with high resolution and a relatively high PK activity, we were able to increase the detection sensitivity of either type 1 or 2, when coexisting, to amount corresponding to 3-5% of the total PrP(Sc) signal (ie, weak band of one type/total PrP(Sc)). Our results show that there are many pitfalls associated with the use of 'type 1 selective' antibodies for CJD typing studies and that co-occurrence of PrP(Sc) types in CJD is not the rule. The present results further validate the rationale basis of current CJD classification and the qualitative nature of molecular typing in CJD.

Bacterial DNA containing CpG motifs activates cells of the innate immune system. In this study, we examined the effects of multiple peripheral bacterial DNA-mediated CNS innate immune stimulation. To study this issue, we repeatedly peripherally administered synthetic CpG-oligodeoxynucleotides (CpG-ODN) and assayed effects on CNS-associated TNF-alpha (TNFalpha) and C1q mRNA levels. We for the first time accounted for frequency of CpG-ODN administration and time kinetics of mRNA expression. We were able show that multiple intraperitoneal CpG-ODN administrations have a sustainable effect on immune effectors of the brain and stimulate TNFalpha mRNA secretion even up to 7 days after the last CpG-ODN application. This could on the one hand indicate a depot effect after multiple peripheral CpG-ODN administrations, however, it could also indicate that the cell producing TNFalpha mRNA remains activated for the indicated time period. Furthermore, elevated mRNA levels of C1q were observed, possibly indicating microglial activation after multiple peripheral bacterial DNA administrations. In this study, we have correlated frequency of CpG-ODN administrations with CNS-associated TNFalpha mRNA levels and show that multiple peripheral administrations of CpG-ODN lead to a sustained level of a Th1-associated cytokine in the brain. These findings indicate that the repeated peripherial administration of CpG oligodeoxynucleotides offer a therapeutical possibility for CNS-associated infections and tumors.

Corticobasal degeneration (CBD) is a neurodegenerative disorder affecting movement and cognition, definitively diagnosed only at autopsy. Here, we conduct a genome-wide association study (GWAS) in CBD cases (n=152) and 3,311 controls, and 67 CBD cases and 439 controls in a replication stage. Associations with meta-analysis were 17q21 at MAPT (P=1.42 × 10−12), 8p12 at lnc-KIF13B-1, a long non-coding RNA (rs643472; P=3.41 × 10−8), and 2p22 at SOS1 (rs963731; P=1.76 × 10−7). Testing for association of CBD with top progressive supranuclear palsy (PSP) GWAS single-nucleotide polymorphisms (SNPs) identified associations at MOBP (3p22; rs1768208; P=2.07 × 10−7) and MAPT H1c (17q21; rs242557; P=7.91 × 10−6). We previously reported SNP/transcript level associations with rs8070723/MAPT, rs242557/MAPT, and rs1768208/MOBP and herein identified association with rs963731/SOS1. We identify new CBD susceptibility loci and show that CBD and PSP share a genetic risk factor other than MAPT at 3p22 MOBP (myelin-associated oligodendrocyte basic protein)., Corticobasal degeneration is a rare neurodegenerative disorder that can only be definitively diagnosed by autopsy. Here, Kouri et al. conduct a genome-wide-association study and identify two genetic susceptibility loci 17q21 (MAPT) and 3p12 (MOBP), and a novel susceptibility locus at 8p12.

The BrainNet Europe consortium assessed the reproducibility in the assignment of the type of frontotemporal lobar degeneration (FTLD) with TAR DNA-binding protein (TDP) 43 following current recommendations. The agreement rates were influenced by the immunohistochemical (IHC) method and by the classification strategy followed. p62-IHC staining yielded good uniform quality of stains, but the most reliable results were obtained implementing specific Abs directed against the hallmark protein TDP43. Both assessment of the type and the extent of lesions were influenced by the Abs and by the quality of stain. Assessment of the extent of the lesions yielded poor results repeatedly; thus, the extent of pathology should not be used in diagnostic consensus criteria. Whilst 31 neuropathologists typed 30 FTLD-TDP cases, inter-rater agreement ranged from 19 to 100 per cent, being highest when applying phosphorylated TDP43/IHC. The agreement was highest when designating Type C or Type A/B. In contrast, there was a poor agreement when attempting to separate Type A or Type B FTLD-TDP. In conclusion, we can expect that neuropathologist, independent of his/her familiarity with FTLD-TDP pathology, can identify a TDP43-positive FTLD case. The goal should be to state a Type (A, B, C, D) or a mixture of Types (A/B, A/C or B/C). Neuropathologists, other clinicians and researchers should be aware of the pitfalls whilst doing so. Agreement can be reached in an inter-laboratory setting regarding Type C cases with thick and long neurites, whereas the differentiation between Types A and B may be more troublesome.

Accurate simulations of non-stationary processes in steam turbines by means of Computational Fluid Dynamics (CFD) require precise and extremely fast algorithms for computing real fluid properties. To fulfill these requirements, the International Association for the Properties of Water and Steam (IAPWS) issues the “Guideline on the Fast Calculation of Steam and Water Properties with the Spline-Based Table Look-Up Method (SBTL)” as an international standard. Through the use of this method, spline functions for the independent variables specific volume and specific internal energy (v,u) are generated for water and steam based on the industrial formulation IAPWS-IF97. With these spline functions, thermodynamic and transport properties can be computed. The desired backward functions of the variables pressure and specific volume (p,v), and specific internal energy and specific entropy (u,s) are numerically consistent with the spline functions from (v,u). The properties calculated from these SBTL functions are in agreement with those of IAPWS-IF97 within a maximum relative deviation of 10 to 100 ppm depending on the property and the range of thermodynamic states spanned under the given conditions (range of state). Consequently, the differences between the results of process simulations using the SBTL method and those obtained through the use of IAPWS-IF97 are negligible. Moreover, the computations from the (v,u) spline functions are more than 200 times faster than the iterative calculations with IAPWS-IF97. In order to demonstrate the efficiency and applicability of the SBTL method, the SBTL functions have been implemented into the CFD software TRACE, developed by the German Aerospace Center (DLR). As a result, the computing times required for the simulations of steam flow in a turbine cascade considering real fluid behavior are reduced by a factor of 6–10 in comparison to the calculations based on IAPWS-IF97. Furthermore, computing times are increased by a factor of 1.4 only with respect to CFD calculations where steam is considered to be an ideal gas, through the use of the SBTL method.

Presenilin 1 (PSEN1) mutations are the major cause of autosomal dominant Alzheimer's disease (ADAD). Here we report three novel PSEN1 mutations: Ile238_Lys239insIle, Ala246Pro and Ala164Val from patients who manifested in their twenties, forties and seventies, respectively, with variant clinical presentations of dementia. These cases exemplify the tremendous heterogeneity of clinical phenotypes and age of onset associated with PSEN1 mutations. The possibility of ADAD--not previously suspected in two of our patients--should always be considered in neurodegenerative conditions albeit they might neither exhibit the typical clinical picture of Alzheimer's disease nor early onset dementia, which is regarded the primary clinical sign of hereditary neurodegeneration.

Prion propagation has been modeled in vitro ; however, the low infectious titer of PrP Sc thus generated has cast doubt on the “protein-only” hypothesis. Here we show that prion delivery on suitable nitrocellulose carrier particles abrogates the apparent dissociation of PrP Sc and infectivity. Misfolded prion protein generated by protein misfolding cyclic amplification is as infectious as authentic brain-derived PrP Sc provided that confounding effects related to differences in the size distribution of prion protein aggregates generated in vitro and consecutive differences in regard to biological clearance are abolished.

Ubiquitin-positive, tau- and α-synuclein–negative inclusions are hallmarks of frontotemporal lobar degeneration with ubiquitin-positive inclusions and amyotrophic lateral sclerosis. Although the identity of the ubiquitinated protein specific to either disorder was unknown, we showed that TDP-43 is the major disease protein in both disorders. Pathologic TDP-43 was hyper-phosphorylated, ubiquitinated, and cleaved to generate C-terminal fragments and was recovered only from affected central nervous system regions, including hippocampus, neocortex, and spinal cord. TDP-43 represents the common pathologic substrate linking these neurodegenerative disorders.

Aberrant folding and fibrillar aggregation by polyglutamine (polyQ) expansion proteins are associated with cytotoxicity in Huntington's disease and other neurodegenerative disorders. Hsp70 chaperones have an inhibitory effect on fibril formation and can alleviate polyQ cytotoxicity. Here we show that the cytosolic chaperonin, TRiC, functions synergistically with Hsp70 in this process and is limiting in suppressing polyQ toxicity in a yeast model. In vitro reconstitution experiments revealed that TRiC, in cooperation with the Hsp70 system, promotes the assembly of polyQ-expanded fragments of huntingtin (Htt) into soluble oligomers of approximately 500 kDa. Similar oligomers were observed in yeast cells upon TRiC overexpression and were found to be benign, in contrast to conformationally distinct Htt oligomers of approximately 200 kDa, which accumulated at normal TRiC levels and correlated with inhibition of cell growth. We suggest that TRiC cooperates with the Hsp70 system as a key component in the cellular defense against amyloid-like protein misfolding.

Assessment of Alzheimer's disease (AD)-related neurofibrillary pathology requires a procedure that permits a sufficient differentiation between initial, intermediate, and late stages. The gradual deposition of a hyperphosphorylated tau protein within select neuronal types in specific nuclei or areas is central to the disease process. The staging of AD-related neurofibrillary pathology originally described in 1991 was performed on unconventionally thick sections (100 mum) using a modern silver technique and reflected the progress of the disease process based chiefly on the topographic expansion of the lesions. To better meet the demands of routine laboratories this procedure is revised here by adapting tissue selection and processing to the needs of paraffin-embedded sections (5-15 mum) and by introducing a robust immunoreaction (AT8) for hyperphosphorylated tau protein that can be processed on an automated basis. It is anticipated that this revised methodological protocol will enable a more uniform application of the staging procedure.

This interlaboratory study evaluated the reproducibility of the assessments of neuritic plaques and neurofibrillary tangles (NFTs)-the hallmark lesions of Alzheimer disease-and compared the staining between the BrainNet Europe centers. To reduce the topography-related inconsistencies in assessments, we used a 2-mm tissue microarray (TMA) technique. The TMA block included 42 core samples taken from 21 paraffin blocks. The assessments were done on Bielschowsky and Gallyas silver stains using an immunohistochemical (IHC) method with antibodies directed to beta-amyloid (IHC/Aβ) and hyperphosphorylated tau (IHC/HPtau). The staining quality and the assessments differed between the participants, being most diverse with Bielschowsky (good/acceptable stain in 53% of centers) followed by Gallyas (good/acceptable stain in 57%) and IHC/Aβ (good/acceptable stain in 71%). The most uniform staining quality and assessment was obtained with the IHC/HPtau method (good/acceptable stain in 94% of centers). The neuropathologic diagnostic protocol (Consortium to Establish a Registry for Alzheimer Disease, Braak and Braak, and the National Institute of Aging and Reagan [NIA-Reagan] Institute) that was used significantly influenced the agreement, being highest with NIA-Reagan (54%) recommendations. This agreement was improved by visualization of NFTs using the IHC/HPtau method. Therefore, the IHC/HPtau methodology to visualize NFTs and neuropil threads should be considered as a method of choice in a future diagnostic protocol for Alzheimer disease. Copyright © 2006 by the American Association of Neuropathologists, Inc.

Previous neurophysiological studies on prion protein deficient (Prnp–/–) mice have revealed a significant reduction of slow afterhyperpolarization currents (sIAHP) in hippocampal CA1 pyramidal cells. Here we aim to determine whether loss of PrPC. directly affects the potassium channels underlying sIAHP or if sIAHP is indirectly disturbed by altered intracellular Ca2+ fluxes. Patch-clamp measurements and confocal Ca2+ imaging in acute hippocampal slice preparations of Prnp–/– mice compared to littermate control mice revealed a reduced Ca2+ rise in CA1 neurons lacking PrPC following a depolarization protocol known to induce sIAHP. Moreover, we observed a reduced Ca2+ influx via l-type voltage gated calcium channels (VGCCs). No differences were observed in the protein expression of the pore forming α1 subunit of VGCCs Prnp–/– mice. Surprisingly, the β2 subunit, critically involved in the transport of the α1 subunit to the plasma membrane, was found to be up-regulated in knock out hippocampal tissue. On mRNA level however, no differences could be detected for the α1C, D and β2–4 subunits. In conclusion our data support the notion that lack of PrPC. does not directly affect the potassium channels underlying sIAHP, but modulates these channels due to its effect on the intracellular free Ca2+ concentration via a reduced Ca2+ influx through l-type VGCCs.

The amyloid precursor protein (APP) is critical in the pathogenesis of Alzheimer's disease. The question of its normal biological function in neurons, in which it is predominantly located at synapses, is still unclear. Using autaptic cultures of hippocampal neurons, we demonstrate that hippocampal neurons lacking APP show significantly enhanced amplitudes of evoked AMPA- and NMDA-receptor-mediated EPSCs. The size of the readily releasable synaptic vesicle pool was also increased in neurons lacking APP, whereas the release probability was not affected. In addition, the analysis of spontaneous miniature synaptic currents revealed an augmented frequency in neurons lacking APP, whereas the amplitude of miniature synaptic currents was not found to be altered. Together, these findings strongly indicate that lack of APP increases the number of functional synapses. This hypothesis is further supported by morphometric immunohistochemical analysis revealing an increase of synaptophysin-positive puncta per cultured APP knock-out neuron. In conclusion, lack of APP affects synapse formation and transmission in cultured hippocampal neurons.

The misfolded infectious isoform of the prion protein (PrP(Sc)) is thought to replicate in an autocatalytic manner by converting the cellular form (PrP(C)) into its pathogenic folding variant. The similarity in the amino acid sequence of PrP(C) and PrP(Sc) influences the conversion efficiency and is considered as the major determinant for the species barrier. We performed in vitro conversion reactions on wild-type and mutated PrP(C) to determine the role of the primary sequence for the high susceptibility of bank voles to scrapie. Different conversion efficiencies obtained with bank vole and mouse PrP(C) in reactions with several prion strains were due to differences at amino acid residues 155 and 170. However, the conversion efficiencies obtained with mouse and vole PrP(C) in reactions with sheep scrapie did not correlate with the susceptibility of the respective species to this prion strain. This discrepancy between in vitro and in vivo data may indicate that at least in the case of scrapie transmission to bank voles additional host factors can strongly modulate the species barrier. Furthermore, in vitro conversion reactions with different prion strains revealed that the degree of alteration of the conversion efficiency induced by amino acid exchanges was varying according to the prion strain. These results support the assumption that the repertoire of conformations adopted by a certain PrP(C) primary sequence is decisive for its convertibility to the strain-specific PrP(Sc) conformation.

Prion proteins play a central role in transmission and pathogenesis of transmissible spongiform encephalopathies. The cellular prion protein (PrP(C)), whose physiological function remains elusive, is anchored to the surface of a variety of cell types including neurons and cells of the lymphoreticular system. In this study, we investigated the response of a mouse monocyte/macrophage cell line to exposure with PrP(C) fusion proteins synthesized with a human Fc-tag. PrP(C) fusion proteins showed an attachment to the surface of monocyte/macrophages in nanomolar concentrations. This was accompanied by an increase of cellular tyrosine phosphorylation as a result of activated signaling pathways. Detailed investigations exhibited activation of downstream pathways through a stimulation with PrP fusion proteins, which include phosphorylation of ERK(1,2) and Akt kinase. Macrophages opsonize and present antigenic structures, contact lymphocytes, and deliver cytokines. The findings reported here may become the basis of understanding the molecular function of PrP(C) in monocytes and macrophages.

In modeling advanced steam power cycles, thermodynamic properties as functions of pressure and enthalpy (p,h) or pressure and entropy (p,s) are required in the critical and supercritical regions (region 3 of IAPWS-IF97). With IAPWS-IF97, these calculations require cumbersome two-dimensional iteration of temperature T and specific volume v from (p,h) or (p,s). While these calculations in region 3 are not frequently required, the computing time can be significant. Therefore, the International Association for the Properties of Water and Steam (IAPWS) adopted backward equations for T(p,h), v(p,h), T(p,s), and v(p,s) in region 3, along with boundary equations for the saturation pressure as a function of enthalpy, p3sat(h), and of entropy, p3sat(s). Using the new equations, two-dimensional iteration can be avoided. The numerical consistency of temperature and specific volume obtained in this way is sufficient for most uses. This paper summarizes the need and the requirements for these equations and gives complete numerical information. In addition, numerical consistency and computational speed are discussed.

Western blotting is one powerful research method to specifically detect proteins. However, it has been barely possible to investigate microscopic volumes of tissue so far because of the required minimum volumes and the pretreatment. Herein, we describe a method of performing Western blots directly from the histological section of frozen or paraffin-embedded tissue. Small histological areas of a mouse brain were lysed by section lysis buffer, subjected to a miniaturized SDS-PAGE, and detected by immunoblotting. Thereby, an area equivalent to only 15 cortical neurons of mouse cortex was detectable. This offers the possibility of correlating histological findings to biochemical investigations. In addition, enzymatic pretreatment was applied to identify the glycosylation of the major cleavage product of the prion protein. Moreover, the section lysis buffer is a sophisticated method to conserve and investigate phosphorylation sites as demonstrated here by phopsphorylated Akt and ERK. The presented technique combines histology with Western blotting techniques and will be of value for investigations of discrete tissue areas. (J Histochem Cytochem 54:559-565, 2006)

To study the potential diagnostic value of abnormally phosphorylated tau protein in Creutzfeldt-Jakob disease (CJD) compared to Alzheimer's disease (AD), we determined levels of tau phosphorylated at threonine 231 (p-tau231) and of total tau (t-tau) in cerebrospinal fluid (CSF) of CJD patients, AD patients, and healthy controls (HC). CJD patients showed excessively high t-tau levels but relatively low p-tau(231) concentrations compared to the AD group. t-tau alone yielded the best diagnostic accuracy to differentiate between CJD and AD patients, when compared to p-tau231 and the p-tau231/t-tau ratio (97, 78, and 95% correctly allocated cases, respectively). Our findings indicate a dissociation in the direction of change in CSF levels of t-tau and p-tau231 in CJD when compared to AD.

Prion diseases are caused by a unique type of infectious agent, which is thought to consist of a misfolded beta-sheeted form of the alpha-helical cellular prion protein (PrPC). This misfolded isoform (PrPSc) tends to form insoluble amyloid-like aggregates, impeding classical structural analysis by X-ray crystallography or NMR. Intermolecular crosslinking may provide a means of stabilizing notoriously elusive oligomers for further analysis and may be used for analyzing aggregate architecture by characterising intermolecular contact sites. Using a photo-induced crosslinking method (PICUP), aggregates of recombinant PrP (rPrP) and PrPSc were linked at interacting surfaces via amino acid side chains. The degree of crosslinking within PrP aggregates was adjustable using varying light intensities and could efficiently be monitored by fluorescence correlation spectroscopy. Specific intermolecular crosslinking of PrPSc molecules was achieved even in crude brain homogenate. Functional studies showed that stabilized aggregates of rPrP did not loose their capacity to induce further protein aggregation and crosslinking of PrPSc did not alter significantly the level of infectivity, indicating that photo-induced covalent linkage of PrPSc does not destruct surfaces important for prion propagation.

The optical transmission loss behavior was investigated for commercially available poly(methyl methacrylate) (PMMA) based polymer optical fibers (POFs). POFs were exposed to various climates of temperature and humidity. Optical transmission measurements using multiplexer (a prototype device) reveal that POFs exhibited an early drop-off followed by a slow decline of transmission at 100°C with low humidity and nearly 100% loss of transmission at the early stages of exposure at 92°C with 95% relative humidity (RH) and at 120°C with low humidity. Fourier transform infrared (FTIR) and gel permeation chromatography (GPC) analysis data show no significant molecular changes in the PMMA core after climatic exposures. However, the attenuated total reflection (ATR)–FTIR data shows a few molecular changes in claddings due to degradation. Scanning electron microscopy (SEM) data illustrate the shrinkage and folding structure in claddings. The loss of the optical transmission at the early (initial) stages of exposure is attributed to the physical changes (like thermal expansion), and the same at the later stages mainly to chemical changes (e.g., oxidative degradation). The experiments conducted here show that the POFs optical transmission stability is strongly dependent on the chemical composition of claddings. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 103: 860–870, 2007

Brain banks manage and store fully clinically and pathologically characterised brains. The diversity of techniques used in research projects increases. These biological resource centres are made to adapt brain tissue processing. Furthermore, the development of more sensitive techniques to analyse nucleic acids and proteins offers new fields of exploration when combined with laser capture microdissection in order to decipher the physiopathology of diseases at the cell level. In this study, our goal was to evaluate procedures and set a workflow compatible with the constraints of brain banks, from brain sampling to laser capture microdissection and pre-analytical quality assessment. We compared various methods of freezing brain tissue, focused on morphological quality preservation of brain microscopical structures and on the quality of nucleic acid or protein yields. Staining protocols combined with strategies to lower neurones autofluorescence were adapted for the same purpose. Finally, we found that laser capture microdissection is possible in the setting of brain banks. However, the entire process has to be envisioned from the autopsy to the analysis. The impact on protein or nucleic acid quality is a limitation that restricts the amount of samples available for this purpose.

Background: Recently, six molecular subtypes of sporadic CJD (sCJD) have been identified showing differences regarding the disease course, neuropathologic lesion patterns, and sensitivity to diagnostic tools. Only isolated cases of the rare VV1 type have been reported so far. Objective: To describe the clinical characteristics and neuropathologic lesion profiles in nine cases. Methods: In the years 1993 until late 2003, 571 definite neuropathologically confirmed cases of sporadic CJD were identified in Germany. Of these, nine were homozygous for valine and displayed type 1 of the pathologic PrP Sc in the brain (VV1 type). Results: The authors describe eight men and one woman belonging to the VV1 type. All patients were relatively young at disease onset (median 44 years vs 65 years in all sCJD) with prolonged disease duration (median 21 months vs 6 months in all sCJD). During the initial stages, their main clinical signs were personality changes and slowly progressive dementia as well as focal neurologic deficits. None of the nine VV1 patients had periodic sharp-wave complexes (PSWCs) in the EEG. Only two out of seven displayed the typical signal increase of the basal ganglia on MRI, whereas signal increase of the cortex was seen in all patients. The 14-3-3 protein levels were elevated in CSF in all cases tested. Conclusions: The clinical diagnosis of the VV1 type of sCJD can be best supported by the 14-3-3 test and cortical signal increase on MRI. Because of the young age at onset vCJD is sometimes suspected as a differential diagnosis. MRI plays an important role in differentiating these two disease types and should be performed early during the disease course.

Mutations in the tau gene cause familial frontotemporal dementia with parkinsonism linked to chromosome 17 characterized by filamentous tau protein deposits. Here we describe the clinical and neuropathological features of a case from a newly identified family with an intron 10+3-splice site mutation in the tau gene. The proband presented with severe personality changes and stereotyped speech followed by parkinsonian symptoms. He died at age 56 after a disease duration of approximately 6 years. At autopsy, there was marked frontotemporal degeneration with abundant tau-immunoreactive neuronal and glial inclusions widespread in the cortex and brainstem. RT-PCR analysis revealed a 3.7-fold increase of tau transcripts with exon 10, resulting in an 1.7-fold higher expression level of 4-repeat tau isoforms in soluble tau fractions when compared to control brains and exclusively 4-repeat tau isoforms in the sarcosyl-insoluble tau fractions. In accordance with the hypothesis that the overexpression leads to saturation of microtubule binding sites and an increase of unbound 4-repeat tau isoforms which assemble into filaments, the neuronal and glial inclusions in this case were exclusively composed of 4-repeat tau isoforms. The clinical and neuropathological data of this family are compared with results from the two other published families with the intron 10 + 3 mutation, the MSTD and the SOT 254 family.

Conformational changes and aggregation of specific proteins are hallmarks of a number of diseases, like Alzheimer's disease, Parkinson's disease, and prion diseases. In the case of prion diseases, the prion protein (PrP), a neuronal glycoprotein, undergoes a conformational change from the normal, mainly alpha-helical conformation to a disease-associated, mainly beta-sheeted scrapie isoform (PrP Sc ), which forms amyloid aggregates. This conversion, which is crucial for disease progression, depends on direct PrP C /PrP Sc interaction. We developed a high-throughput assay based on scanning for intensely fluorescent targets (SIFT) for the identification of drugs which interfere with this interaction at the molecular level. Screening of a library of 10,000 drug-like compounds yielded 256 primary hits, 80 of which were confirmed by dose response curves with half-maximal inhibitory effects ranging from 0.3 to 60 μM. Among these, six compounds displayed an inhibitory effect on PrP Sc propagation in scrapie-infected N2a cells. Four of these candidate drugs share an N ′-benzylidene-benzohydrazide core structure. Thus, the combination of high-throughput in vitro assay with the established cell culture system provides a rapid and efficient method to identify new antiprion drugs, which corroborates that interaction of PrP C and PrP Sc is a crucial molecular step in the propagation of prions. Moreover, SIFT-based screening may facilitate the search for drugs against other diseases linked to protein aggregation.

Background: An international study of the epidemiologic characteristics of Creutzfeldt–Jakob disease (CJD) was established in 1993 and included national registries in France, Germany, Italy, the Netherlands, Slovakia, and the United Kingdom. In 1997, the study was extended to Australia, Austria, Canada, Spain, and Switzerland. Methods: Data were pooled from all participating countries for the years 1993 to 2002 and included deaths from definite or probable CJD of all etiologic subtypes. Results: Four thousand four hundred forty-one cases were available for analysis and included 3,720 cases of sporadic CJD, 455 genetic cases, 138 iatrogenic cases, and 128 variant cases. The overall annual mortality rate between 1999 and 2002 was 1.67 per million for all cases and 1.39 per million for sporadic CJD. Mortality rates were similar in all countries. There was heterogeneity in the distribution of cases by etiologic subtype with an excess of genetic cases in Italy and Slovakia, of iatrogenic cases in France and the UK, and of variant CJD in the UK. Conclusions: This study has established overall epidemiologic characteristics for Creutzfeldt–Jakob disease (CJD) of all types in a multinational population–based study. Intercountry comparisons did not suggest any relative change in the characteristics of sporadic CJD in the United Kingdom, and the evidence in this study does not suggest the occurrence of a novel form of human bovine spongiform encephalopathy infection other than variant CJD. However, this remains a possibility, and countries currently unaffected by variant CJD may yet have cases.

Prion diseases are characterized by the conversion of the cellular prion protein (PrP(C)) to a disease-specific aggregated isoform (PrP(Sc)). We have shown that Mn(2+) ions amplify aggregation, whereas Cu(2+) has an inhibitory effect. To characterize Mn(2+)-induced aggregates, we used cross-correlation analysis as well as scanning for intensely fluorescent targets in an SDS-dependent aggregation assay with fluorescently labeled PrP. We found that the effect of Mn(2+) was mainly due to the association of preformed PrP oligomers to larger aggregates, rapidly reversible by EDTA, and independent of the histidine-dependent copper-binding sites of PrP, suggesting that Mn(2+) induces reversible intermolecular binding. In contrast, the inhibitory effect of Cu(2+) required binding to histidine-containing binding sites, indicating that binding of copper affects the structure of PrP(C) which in turn modifies the susceptibility to manganese and the ability to aggregate. These findings suggest that copper and manganese may also affect prion propagation in vivo.

Sirs, Prion diseases are transmissible diseases that include sporadic, acquired and familial disorders. More than 30 point mutations and insert mutations of the prion protein gene (PRNP) are thought to cause familial prion diseases, i.e. familial Creutzfeldt-Jakob disease (fCJD), GerstmannStraussler-Scheinker syndrome (GSS) and fatal familial insomnia (FFI). Clinical and pathological changes may be similar or indistinguishable from sporadic CJD (sCJD) as in fCJD associated with the E200K mutation, or may be quite different from sCJD as in the P102L mutation that is associated with GSS [1]. The clinical and pathological appearance of sCJD is linked to a methionine–valine polymorphism at codon 129 of PRNP and the biochemical isoform of PrP [2]. The influence of the codon 129 polymorphism in familial prion diseases depends on the particular pathogenic mutation and can be significant. Here we report on a CJD case with an R148H mutation. This patient underwent surgery for hysterectomy when she was 82 years old. After surgery she recovered, but was found somewhat fearful. Five months later she developed a rapidly progressive depressive, anxious disorder, and her state of awareness deteriorated within two weeks. She was admitted to a psychiatric ward. On admission she was disoriented, apprehensive, mistrustful and mute; Babinski’s sign on the right was positive. Within days the patient became somnolent and reacted with a defence reaction to painful stimuli. An evident ‘gegenhalten’ of all extremities was found, and the patient developed myoclonic jerks. She had to be fed by tube. The first EEG showed generalized pathologic alterations, but later periodic sharp wave complexes appeared. In the CT mild cortical atrophy was seen. T2-weighted MRI images revealed symmetric multiple hyperintense lesions in subcortical and periventricular locations. Examinations of the CSF was positive for the 143-3 protein and showed an increased tau protein level (>1,500 pg/ml). The patient was diagnosed with probable sCJD [3]. There was only limited information on her family, but no disease similar to CJD was known in the family. She succumbed six months after the onset of disease and an autopsy was performed. Genomic DNAwas extracted from peripheral blood and was investigated as described [4]. For Western blotting the brain was kept frozen at −80°C until use. Tissue from the frontal cortex and cerebellum was homogenised in 9 volumes (wt./vol) of lysis buffer (0.5% Nonidet P-40, 0.5% DOC, 10 mM EDTA, 100 mM NaCl, 100 mM Tris, pH 7.4) and further processed as described [2]. After PK digestion (50 μg/ml, 1 h at 37°C) cleared homogenates were subjected to SDS-PAGE and blotted on PVDF membranes. Monoclonal antibodies 6H4, 3F4 and L42 were used for immunodetection [5–7]. Blots were developed using NBT-BCIP according to standard protocols. Quantification of band intensities was performed with software TotalLab 1D Gel Analysis, version 2.0. For microscopic investigation the right half of the brain was fixed in formalin and processed for histology, immunohistochemistry and paraffin-embedded tissue (PET) blotting [8]. The monoclonal antibodies L42 and 12F10 were used for immunohistochemistry and PET blotting respectively. B. Krebs . R.-M. Lederer . O. Windl . E.-M. Grasbon-Frodl . H. A. Kretzschmar (*) Institut fur Neuropathologie, LMU Munchen, Feodor-Lynen-Strasse 23, 81377 Munich, Germany e-mail: Hans.Kretzschmar@med.uni-muenchen.de

Mutations in the tau gene cause familial frontotemporal dementia and parkinsonism linked to chromosome 17. Here we describe a novel missense mutation in exon 12 of the tau gene, G335V, in a German family with frontotemporal dementia of early age at onset, in the third decade of life. Functional analysis of recombinant tau protein with the G335V mutation showed a dramatically reduced ability to promote microtubule assembly and a more rapid and accelerated tau filament formation, suggesting that the primary effect of the mutation might be the provision of a pool of unbound tau making it available for aberrant tau aggregation.

Objectives – Cerebrospinal fluid (CSF) pattern in patients with neuropathologically diagnosed Creutzfeldt–Jakob disease was analyzed. Material and methods – Routine tests included white blood cells count, protein, albumin, immunoglobulins and the presence of oligoclonal immunoglobulin G (IgG) in the CSF as well as the calculation of intrathecal synthesis of immunoglobulins by standard methods. In addition, antibodies against neurotropic viruses such as measles, rubella, varicella zoster and herpes simplex were measured and the specific antibody index was calculated. Results – A blood–CSF barrier dysfunction was observed in six of 25 cases. In CSF/serum quotient diagrams, no patient had intrathecally synthesized immunoglobulins, but in two of 25 patients oligoclonal bands were detected. Two patients had intrathecally synthesized antibodies against varicella zoster and three against herpes simplex virus. Conclusion – In conclusion, in the routine diagnosis, the CSF in CJD is normal in most cases. In some patients, abnormalities include the blood–CSF barrier dysfunction, mild pleocytosis, oligoclonal bands and intrathecally synthesized viral antibodies.

A case of Creutzfeldt-Jakob disease (CJD) with a rare mutation of the prion protein (PrP) gene (PRNP) at codon 208 (R208H) is described. By comparison with two preceding reports, the case described here displayed two distinct biochemical and neuropathological features. Western blot analysis of brain homogenates showed, in addition to the commonly observed three bands of abnormal protease-resistant PrP isoform (PrP(Sc)), an additional band of about 17 kDa. Neuropathological examination of the post mortem brain revealed tau pathology in the hippocampus and entorhinal cortex, as well as ballooned neurons in the cortex, hippocampus and subcortical gray matter.

Oligodeoxynucleotides containing CpG motifs (CpG-ODN) activate cells of the innate immune system. Recent studies have shown that sole CpG-ODN administration induces resistance against infection and tumors. Effects of CpG-ODN administration are rapidly induced, and regarding infections only short-term protection was seen. One conceivable strategy to prolong protective effects is multiple administrations of CpG-ODN. However, inappropriate immune activation via CpG motifs has been implicated in septic shock and autoimmunity. To investigate effects of multiple CpG-ODN administrations, we analyzed Th1- and Th-2-associated Ig antibody levels, during and after multiple treatment with CpG-ODN. Our results show that multiple administrations of CpG-ODN lead to an increase in total IgG2c levels in CpG-ODN-treated mice in comparison to controls with distinct time and frequency correlation, in the absence of additional stimuli. This indicates a humoral Th1 bias based on stimulation of Th1-Ig isotype-producing B cells. These effects could account for observed anti-infection and anti-tumor properties of multiple CpG-ODN administrations; on the other hand, they might cause autoimmune disease.

The conversion of cellular prion protein (PrP(C)) to the disease-associated misfolded isoform (PrP(Sc)) is an essential process for prion replication. This structural conversion can be modelled in protein misfolding cyclic amplification (PMCA) reactions in which PrP(Sc) is inoculated into healthy hamster brain homogenate, followed by cycles of incubation and sonication. In serial transmission PMCA experiments it has recently been shown that the protease-resistant PrP obtained in vitro (PrPres) is generated by an autocatalytic mechanism. Here, serial transmission PMCA experiments were compared with serial transmission reactions lacking the sonication steps. We achieved approximately 200,000-fold PrPres amplification by PMCA. In contrast, although initial amplification was comparable to PMCA reactions, PrPres levels quickly dropped below detection limit when samples were not subjected to ultrasound. These results indicate that aggregate breakage is essential for efficient autocatalytic amplification of misfolded prion protein and suggest an important role of aggregate breakage in prion propagation.

The prion protein (PrPC) is a copper-binding, cell-surface protein that plays an essential role in the etiology of transmissible spongiform encephalopathies. Atomic absorption spectroscopy studies have established that synaptosomal copper content is reduced in PrPC-deficient mice as compared with wild-type (WT) or PrP- overexpressing mice. To address the question of whether this is the result of a loss of function of PrPC in copper transport across the plasma membrane at the synapse, we have used synaptosomes incubated with 67Cu as a model system to characterize the mechanism of copper accumulation in nerve terminals. Our results demonstrate that mouse brain synaptosomes accumulate copper efficiently by at least two distinct mechanisms. In the presence of 1 mM EDTA, copper was taken up via a saturable high-affinity process that was moderately susceptible to competition by high concentrations of NiCl2. Uptake characteristics were clearly different in the presence of 400 microM histidine, with the most noticeable dissimilarities being considerably elevated uptake rates and moderate competition by ZnCl2 rather than NiCl2. No significant differences in copper uptake capability between WT and PrPC-knockout synaptosomes were observed under any of the experimental conditions tested in this study. Furthermore, preincubation of synaptosomes with an antibody binding to the copper-binding repeat region of the prion protein had no effect on copper uptake either. Taken together, our data indicate that synaptosomal copper uptake is independent of PrPC.

The objective of the study was to identify clinical features that distinguish patients with dementia with Lewy bodies (DLB), who were classified as Alzheimer’s disease (AD) patients, from patients with AD. We examined a group of 27 patients from our memory clinic, originally diagnosed with AD, of whom 6 were postmortem found to have DLB. For the present study, we compared cognitive, noncognitive and neurological symptoms between the two groups. We found that there were no differences on ratings of dementia and scales for activities of daily living. Patients with DLB performed better on the MMSE and the memory subtest of the CAMCOG, but there was no difference in any other cognitive domain. Furthermore, genetic risk factors, including family history of dementia or allele frequency of the apolipoprotein ε4, did not discriminate between the two groups, and there were no differences on CCT scans. Taken together, our findings suggest that Lewy body pathology may be present in patients who do not show the typical clinical features which distinguish DLB from AD.

Sporadic Creutzfeldt-Jakob disease (sCJD) is a rare neurodegenerative disease with the greatest incidence occurring in patients between 60 and 70 years old. Younger patients may also be affected. In this study, we used all case material available from 52 patients with sCJD aged 50 years or younger at disease onset, who were identified between 1993 and 2003 in Germany. The objective of this study was to describe the psychiatric and neurological features of these young patients with emphasis on the different codon 129 genotypes and PrP types, and to compare them with elder patients with sCJD and patients with variant CJD. We also gave particular attention to electroencephalogram, magnetic resonance imaging, and 14-3-3 results, as well as to the neuropathological lesion profile. The clinical syndrome in young patients differs from elder patients with CJD with respect to clinical signs, disease duration, technical investigations, and neuropathological lesion profile. The psychiatric symptoms in young patients with sCJD are similar to the psychiatric symptoms expressed by patients with variant CJD; however, in contrast with the variant cases, young patients with sCJD experience development of prominent dementia early in the disease course.

Progressive multifocal leukoencephalopathy (PML) is a subaute encephalitis caused by the JC polyomavirus almost always in atients with severe cellular immune deficiency, nowadays most requently in patients with human immunodeficiency virus (HIV) nfection or recently as a complication of Natalizumab treatment or Multiple Sclerosis. Occasionally cases in immunocompetent atients have been reported. MRI displays a typical pattern of ultiple subcortical areas of high signal on FLAIR images in both emispheres. Single cases of purely infratentorial involvement ave been reported but are very infrequent [1]. Here we report a ase of primary brainstem involvement of PML in an immunocometent patient with a deleterious course and negative JC-virus but ositive BK-virus PCR in the CSF. The use and findings of PET-MRI re discussed.

The PrP-like Doppel (Dpl) protein causes apoptotic death of cerebellar neurons in transgenic mice, a process prevented by expression of the wild type (wt) cellular prion protein, PrP(C). Internally deleted forms of PrP(C) resembling Dpl such as PrPDelta32-121 produce a similar PrP(C)-sensitive pro-apoptotic phenotype in transgenic mice. Here we demonstrate that these phenotypic attributes of wt Dpl, wt PrP(C), and PrPDelta132-121 can be accurately recapitulated by transfected mouse cerebellar granule cell cultures. This system was then explored by mutagenesis of the co-expressed prion proteins to reveal functional determinants. By this means, neuroprotective activity of wt PrP(C) was shown to be nullified by a deletion of the N-terminal charged region implicated in endocytosis and retrograde axonal transport (PrPDelta23-28), by deletion of all five octarepeats (PrPDelta51-90), or by glycine replacement of four octarepeat histidine residues required for selective binding of copper ions (Prnp"H/G"). In the case of Dpl, overlapping deletions defined a requirement for the gene interval encoding helices B and B' (DplDelta101-125). These data suggest contributions of copper binding and neuronal trafficking to wt PrP(C) function in vivo and place constraints upon current hypotheses to explain Dpl/PrP(C) antagonism by competitive ligand binding. Further implementation of this assay should provide a fuller understanding of the attributes and subcellular localizations required for activity of these enigmatic proteins.

Lewy bodies (LBs) containing alpha-synuclein (alphaSYN) fibrils are the hallmark lesions of Parkinson disease, dementia with LBs, and related neurodegenerative diseases. Here we have investigated the susceptibility to proteolysis of alphaSYN from brain samples of patients with different subtypes of LB diseases. While soluble alphaSYN was completely degradable in all samples, the affected brain regions additionally contained insoluble, proteinase K (PK)-resistant alphaSYN. In brainstem-predominant subtype LB disease cases, PK-resistant alphaSYN was found in the medulla and substantia nigra, but not in cerebral cortex. In limbic subtype LB disease cases, PK resistance of alphaSYN spread to the cingulate and parahippocampal cortex, and further to the frontal cortex in neocortical subtype LB disease cases. Variable amounts of neuritic PK-resistant alphaSYN were found in the striatum of all cases. Thus, PK resistance of alphaSYN may be useful for the development of biomarkers of LB diseases.

Involvement of the interleukin-6 receptor complex (IL-6RC) in neuroregulatory and immunological processes of the brain and particularly in Alzheimer's disease (AD) has been hypothesized. The functionally active IL-6RC consists of the cytokine IL-6, which acts through the ligand binding IL-6R and the signal transducing gp130. Using a new immunocytochemical protocol on rapid autopsy cryostat brain sections we studied the expression of the IL-6RC in Braak IV-V staged AD patients compared to normal age-matched controls (HC) across five different cortical regions. Inter-rater reliability of the method was high. The "baseline" expression in normal human brain was determined for IL-6,IL-6R and gp130 in all cortical regions. In normal tissue IL-6 expression was lower in parietal cortex. Higher IL-6R expression was shown in frontal, occipital and parietal cortex, lower expression in temporal cortex and cerebellum. In AD IL-6 expression levels were generally increased in parietal cortex and decreased in occipital cortex compared to controls. IL-6R expression levels were strongly increased in AD frontal and occipital cortex and decreased in temporal cortex and cerebellum. Our findings indicate an altered cortical immunoreactivity pattern of the functional IL-6RC in AD supporting the hypothesis of a disease-related role of IL-6 in AD pathophysiology.

Measuring proteins in cerebrospinal fluid (CSF) has gained wide acceptance for the differential diagnosis of dementia. Some groups have already extended these investigations in Alzheimer's disease (AD) by asking how stable these markers are in follow-up analysis, if they depend on the stage of disease and whether they can be used to monitor the progression and biological effects of treatment. We evaluated 21 patients with dementia with Lewy bodies (DLB) and 19 patients with AD, on two occasions, with regard to levels of tau protein, tau protein phosphorylated at threonine 181 (p-tau), Abeta42, Abeta40 and S-100B protein, using a set of commercially available assays. Tau protein levels were lower in DLB in first and second LP compared to AD and decreased during course of both groups. P-tau levels were increased in AD and DLB and decreased during follow-up. Abeta42 and Abeta40 remained relatively stable during follow-up but we found a slight increase of the median Abeta42 level in DLB, whereas in AD, Abeta42 tends to decrease during follow-up. S-100B protein increased during follow-up in both diseases. The protein dynamics in DLB and AD are relatively similar. S-100B protein may be a useful marker for follow-up in neurodegenerative diseases but has to be analysed in longer follow-up periods. Tau protein may be used to differentiate between DLB and AD. Follow-up CSF analyses are of limited value for the differentiation of AD and DLB. We conclude that more specific markers have to be established for the differentiation and follow-up of these diseases.

Intravascular lymphomatosis (IVL), a rare type of non-Hodgkin's lymphoma, is an uncommon cause of progressive dementia, usually followed by death within a few months of onset of clinical disease. Often this aggressive tumor is only diagnosed at autopsy, because of misleading clinical features mimicking a broad spectrum of syndromes and the absence of circulating lympoma cells in the blood, bone marrow or cerebrospinal fluid in many cases. Here we present IVL in a 78-year-old woman with findings leading to the clinical diagnosis of vascular dementia with sudden beginning and positive 14-3-3 protein in the CSF, commonly reported in Creutzfeldt-Jakob disease (CJD).

The diagnosis of neurodegenerative diseases with dementias requires several different test approaches and often remains uncertain. Using a proteomic approach it was shown in nine patients that heart fatty acid binding protein (H-FABP) might be a biomarker for Creutzfeldt-Jakob disease (CJD). The aim of our study was to evaluate whether H-FABP is a biomarker for the differential diagnosis of dementias. Therefore we measured H-FABP in cerebrospinal fluid (CSF) and serum of patients having CJD, dementia with Lewy-bodies (DLB), Alzheimer's disease (AD) and in non-demented control (NDC) patients. H-FABP levels in CSF and serum of CJD patients are increased compared to non-demented controls. Levels of H-FABP were significantly higher in CJD patients compared to AD and DLB in CSF. However, discrimination between CJD and AD was not possible in serum. Interestingly, highest levels of H-FABP were found in serum of DLB patients. Our results suggest that H-FABP might be a useful biomarker for the differentiation between the dementias examined if levels in CSF and serum are determined in parallel.

Objective: To assess if clinical features, prion protein codon 129, and molecular subtype correlate with MRI basal ganglia hyperintensity in sporadic Creutzfeldt-Jakob disease (CJD). Methods: The authors studied 219 patients including 153 confirmed CJD cases for their neurologic symptoms and MRI findings. The MRI was assessed by a blinded investigator for the presence of high signal intensity on T2-weighted images in the basal ganglia. Results: Patients with basal ganglia high signal on T2-weighted images were more likely to present with rapid progressive dementia in an early stage and shorter disease duration (median 6.7 months and 8.6 months). Surprisingly, among the CJD cases, patients without signal increase of the basal ganglia were shown to have a higher frequency of extrapyramidal disturbances (82% vs 70%). More striking differences were found for symptoms such as depression and sensory disturbances, which were more frequent among cases without signal increase. MRI was more likely to be diagnostic in patients with MV2 molecular subtype. Conclusions: Selected clinical and pathologic features correlate with the presence of basal ganglia high signal on T2-weighted MRI in patients with definite or probable CJD.

Mutations in the PARK7 gene DJ-1 are associated with recessive hereditary Parkinson's disease (PD). Fibrillar inclusions of alpha-synuclein comprise the neuropathological hallmarks of PD and related Lewy body diseases as well as multiple system atrophy (MSA). Moreover, neuronal and glial inclusions containing tau have been observed in alpha-synucleinopathy patients. Using a collection of antibodies against DJ-1, we have performed a comprehensive investigation of DJ-1 in alpha-synucleinopathies and tauopathies. DJ-1 was abundantly expressed in reactive astrocytes of patients with neurodegenerative diseases. Likewise, DJ-1 antiserum immunostained reactive astrocytes that became abundant with disease progression in the brain stem of transgenic mice expressing mutant [A30P]alpha-synuclein. Human Lewy bodies as well as Lewy body-like inclusions in the alpha-synuclein transgenic mice were DJ-1 negative. Neuronal tau inclusions were DJ-1 immunopositive in Pick's disease (PiD), corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), and Alzheimer's disease. In addition, we found DJ-1-immunopositive glial inclusions in CBD, PSP and MSA. Biochemical extraction experiments revealed the specific presence of insoluble, modified DJ-1 in PiD and MSA. Our results suggest that DJ-1 is up-regulated in reactive astrocytes as well as in neuronal and glial cells with specific alpha-synucleinopathy and tauopathy.

The discovery of molecular subtypes of the pathological prion protein PrPSc has provided the basis for a novel classification of human transmissible spongiform encephalopathies (TSEs) and a potentially powerful method for strain typing. However, there is still a significant disparity regarding the understanding and nomenclature of PrPSc types. In addition, it is still unknown whether a specific PrPSc type is associated with each TSE phenotypic variant. In sporadic Creutzfeldt-Jakob disease (sCJD), five disease phenotypes are known, but only two major types of PrPSc, types 1 and 2, have been consistently reproduced. We further analyzed PrPSc properties in sCJD and variant CJD using a high resolution gel electrophoresis system and varying experimental conditions. We found that pH varies among CJD brain homogenates in standard buffers, thereby influencing the characteristics of protease-treated PrPSc. We also show that PrPSc type 1 and type 2 are heterogeneous species which can be further distinguished into five molecular subtypes that fit the current histopathological classification of sCJD variants. Our results shed light on previous disparities in PrPSc typing, provide a refined classification of human PrPSc types, and support the notion that the pathological TSE phenotype is related to PrPSc structure.

The prion protein (PrP C ) has a primary role in the pathogenesis of transmissible spongiform encephalopathies. Here we analysed in detail the effect of recombinant PrP C and N- and C-terminal fragments of PrP C on the whole-cell current amplitude through voltage-gated calcium channels (VGCCs) of cultured wild-type cerebellar granule cells. With the application of full-length recombinant PrP C (50–500 nM), a highly significant reduction of the whole-cell current amplitude was observed in a dose-dependent manner. Amplitude reduction was abolished when cells were pre-incubated with nifedipine, a specific blocker of voltage-gated L-type calcium channels. N-terminal PrP fragments also led to a dose-dependent reduction of the maximal current amplitude, whereas a C-terminal fragment did not affect the current amplitude. These data demonstrate that nanomolar concentrations of PrP C modulate L-type VGCCs in mouse cerebellar granule cells, an effect that is dependent upon the copper-binding amino-terminal domain of PrP C .

ZusammenfassungUnter dem klinischen Terminus frontotemporale Demenz (FTD) wird eine heterogene Gruppe neurodegenerativer Erkrankungen zusammengefasst. In den vergangenen Jahren sind die Erkenntnisse über genetische Ursachen und molekulare Mechanismen der FTD rasch vorangeschritten und haben einen Wandel der Klassifikation wie auch der Nomenklatur auf diesem Gebiet eingeleitet. Trotz Fortschritten in der klinischen Diagnostik ist eine Unterscheidung der einzelnen FTD-Entitäten derzeit nur durch eine neuropathologische Untersuchung des Gehirns möglich. Die gängige neuropathologische Klassifikation der FTD basiert auf der Morphologie sowie der histochemischen und biochemischen Charakterisierung von Einschlusskörperchen. Die Spezifizierung der Einschlüsse erfolgt hierbei mittels Antikörpern gegen Tau, Ubiquitin und Neurofilamente. Zur weiteren Erforschung der zugrunde liegenden molekularen Mechanismen der FTD insbesondere im Hinblick auf die Entwicklung neuer Therapien, sind Untersuchungen an Hirngewebe von klinisch und neuropathologisch gut dokumentierten Fällen dringend erforderlich. Einen wichtigen Beitrag hierzu können Hirngewebebanken, wie die Deutsche Hirngewebebank »Brain-Net«, leisten.