Prion disease is characterized by the αrightward arrowβ structural conversion of the cellular prion protein (PrPC) into the misfolded and aggregated "scrapie" (PrPSc) isoform. It has been speculated ...that methionine (Met) oxidation in PrPC may have a special role in this process, but has not been detailed and assigned individually to the 9 Met residues of full-length, recombinant human PrPC rhPrPC(23-231). To better understand this oxidative event in PrP aggregation, the extent of periodate-induced Met oxidation was monitored by electrospray ionization-MS and correlated with aggregation propensity. Also, the Met residues were replaced with isosteric and chemically stable, nonoxidizable analogs, i.e., with the more hydrophobic norleucine (Nle) and the highly hydrophilic methoxinine (Mox). The Nle-rhPrPC variant is an α-helix rich protein (like Met-rhPrPC) resistant to oxidation that lacks the in vitro aggregation properties of the parent protein. Conversely, the Mox-rhPrPC variant is a β-sheet rich protein that features strong proaggregation behavior. In contrast to the parent Met-rhPrPC, the Nle/Mox-containing variants are not sensitive to periodate-induced in vitro aggregation. The experimental results fully support a direct correlation of the αrightward arrowβ secondary structure conversion in rhPrPC with the conformational preferences of Met/Nle/Mox residues. Accordingly, sporadic prion and other neurodegenerative diseases, as well as various aging processes, might also be caused by oxidative stress leading to Met oxidation.
The transcription factor Forkhead box M1 (FoxM1) is a key regulator of cell-cycle progression. It is involved in the development of multiple organs, and we have previously reported on its important ...role for the mitotic entry of cerebellar granule neuron precursors. Constitutive expression of FoxM1 is required for the growth of multiple cancer types. This study aimed to determine its role in medulloblastoma, the most frequent malignant brain tumor in childhood that can derive from cerebellar granule neuron precursors.
We evaluated the expression of FoxM1 together with its prognostic value in two independent series of human medulloblastoma samples using immunohistochemistry (n = 43) and gene expression arrays (n = 193). The functional impact of FoxM1 expression was characterized by knockdown experiments in four human medulloblastoma cell lines, and the thiazole antibiotic siomycin A was tested to downregulate FoxM1 and inhibit tumor cell growth.
FoxM1 was highly expressed in all subtypes of medulloblastoma. Importantly, expression levels of FoxM1 significantly correlated with unfavorable clinical outcome in univariate analysis (P = 0.0005), and FoxM1 was identified as an independent prognostic marker by multivariate analysis (P = 0.037). Knockdown of FoxM1 in medulloblastoma cell lines resulted in a significant decrease of cell viability which was caused by a failure in mitotic spindle formation and caspase-dependent mitotic catastrophe. Siomycin A significantly inhibited the expression of FoxM1 and the growth of medulloblastoma cells.
FoxM1 may be used as an additional prognostic marker and may represent a potential novel target to treat patients suffering from medulloblastoma.
Pathologic TAR-DNA-binding protein 43 (TDP-43) is a disease protein in frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis. We studied the ...presence, frequency, and distribution of TDP-43 pathology by immunohistochemistry and biochemistry in a series of clinically well-characterized tauopathy patient brains, including 182 Alzheimer disease (AD), 39 corticobasal degeneration, 77 progressive supranuclear palsy, and 12 Pick disease cases and investigated the clinical impact of concomitant TDP-43 pathology in these cases. TAR-DNA-binding protein 43 pathology was found in 25.8% of AD cases. It was restricted to the dentate gyrus and entorhinal cortex in approximately 75% of cases; approximately 25% showed more widespread TDP-43 pathology in frontal and temporal cortices, resembling the FTLD-U subtype associated with progranulin mutations. TAR-DNA-binding protein 43 pathology in AD was associated with significantly longer disease duration, but there was no association with the clinical presentation (148 cases diagnosed as AD and 34 cases diagnosed as frontotemporal lobar degeneration). Progressive supranuclear palsy and Pick disease cases showed no TDP-43 inclusions and no biochemical alterations of TDP-43. There was, however, a unique, predominantly glial TDP-43 pathology with staining of astrocytic plaque-like structures and coiled bodies in 15.4% of corticobasal degeneration cases; this was associated with biochemical TDP-43 changes similar to those in FTLD-U. These findings provide further insight into the burden and clinical significance of TDP-43 pathology in disorders other than FTLD-U and amyotrophic lateral sclerosis.
5‐Methylcytosine (5mC) in genomic DNA has important epigenetic functions in embryonic development and tumor biology. 5‐Hydroxymethylcytosine (5hmC) is generated from 5mC by the action of the TET ...(Ten‐Eleven‐Translocation) enzymes and may be an intermediate to further oxidation and finally demethylation of 5mC. We have used immunohistochemistry (IHC) and isotope‐based liquid chromatography mass spectrometry (LC‐MS) to investigate the presence and distribution of 5hmC in human brain and brain tumors. In the normal adult brain, IHC identified 61.5% 5hmC positive cells in the cortex and 32.4% 5hmC in white matter (WM) areas. In tumors, positive staining of cells ranged from 1.1% in glioblastomas (GBMs) (WHO Grade IV) to 8.9% in Grade I gliomas (pilocytic astrocytomas). In the normal adult human brain, LC‐MS also showed highest values in cortical areas (1.17% 5hmC/dG deoxyguanosine), in the cerebral WM we measured around 0.70% 5hmC/dG. 5hmC levels were related to tumor differentiation, ranging from lowest values of 0.078% 5hmC/dG in GBMs (WHO Grade IV) to 0.24% 5hmC/dG in WHO Grade II diffuse astrocytomas. 5hmC measurements were unrelated to 5mC values. We find that the number of 5hmC positive cells and the amount of 5hmC/dG in the genome that has been proposed to be related to pluripotency and lineage commitment in embryonic stem cells is also associated with brain tumor differentiation and anaplasia.
Bovine spongiform encephalopathy (BSE) is a fatal, transmissible, neurodegenerative disease of cattle. To date, the disease process is still poorly understood. In this study, brain tissue samples ...from animals naturally infected with BSE were analysed to identify differentially regulated genes using Affymetrix GeneChip Bovine Genome Arrays. A total of 230 genes were shown to be differentially regulated and many of these genes encode proteins involved in immune response, apoptosis, cell adhesion, stress response and transcription. Seventeen genes are associated with the endoplasmic reticulum (ER) and 10 of these 17 genes are involved in stress related responses including ER chaperones, Grp94 and Grp170. Western blotting analysis showed that another ER chaperone, Grp78, was up-regulated in BSE. Up-regulation of these three chaperones strongly suggests the presence of ER stress and the activation of the unfolded protein response (UPR) in BSE. The occurrence of ER stress was also supported by changes in gene expression for cytosolic proteins, such as the chaperone pair of Hsp70 and DnaJ. Many genes associated with the ubiquitin-proteasome pathway and the autophagy-lysosome system were differentially regulated, indicating that both pathways might be activated in response to ER stress. A model is presented to explain the mechanisms of prion neurotoxicity using these ER stress related responses. Clustering analysis showed that the differently regulated genes found from the naturally infected BSE cases could be used to predict the infectious status of the samples experimentally infected with BSE from the previous study and vice versa. Proof-of-principle gene expression biomarkers were found to represent BSE using 10 genes with 94% sensitivity and 87% specificity.
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).
Objective
Amyotrophic lateral sclerosis (ALS) is a common, fatal motor neuron disorder with no effective treatment. Approximately 10% of cases are familial ALS (FALS), and the most common genetic ...abnormality is superoxide dismutase‐1 (SOD1) mutations. Most ALS research in the past decade has focused on the neurotoxicity of mutant SOD1, and this knowledge has directed therapeutic strategies. We recently identified TDP‐43 as the major pathological protein in sporadic ALS. In this study, we investigated TDP‐43 in a larger series of ALS cases (n = 111), including familial cases with and without SOD1 mutations.
Methods
Ubiquitin and TDP‐43 immunohistochemistry was performed on postmortem tissue from sporadic ALS (n = 59), ALS with SOD1 mutations (n = 15), SOD‐1–negative FALS (n = 11), and ALS with dementia (n = 26). Biochemical analysis was performed on representative cases from each group.
Results
All cases of sporadic ALS, ALS with dementia, and SOD1‐negative FALS had neuronal and glial inclusions that were immunoreactive for both ubiquitin and TDP‐43. Cases with SOD1 mutations had ubiquitin‐positive neuronal inclusions; however, no cases were immunoreactive for TDP‐43. Biochemical analysis of postmortem tissue from sporadic ALS and SOD1‐negative FALS demonstrated pathological forms of TDP‐43 that were absent in cases with SOD1 mutations.
Interpretation
These findings implicate pathological TDP‐43 in the pathogenesis of sporadic ALS. In contrast, the absence of pathological TDP‐43 in cases with SOD1 mutations implies that motor neuron degeneration in these cases may result from a different mechanism, and that cases with SOD1 mutations may not be the familial counterpart of sporadic ALS. Ann Neurol 2007;61:427–434
In 1996, our group published objective electroencephalogram (EEG) criteria to define periodic sharp‐wave complexes (PSWCs) suggestive for Creutzfeldt–Jakob disease (CJD). These criteria have since ...then been strictly applied in all cases reported to us as possible CJD in the course of the German CJD surveillance study. Furthermore, EEG analysis of the records was performed without any additional information on complementary clinical and laboratory data. In this study, we investigated sensitivity, specificity, and the predictive values of these EEG criteria exclusively in cases in which autopsy confirmed (n = 150) or excluded (n = 56) CJD. EEG criteria were positive in 64% (n = 96) of the CJD cases and falsely positive in 9% (n = 5) of other dementias. The resulting figures for sensitivity, specificity, and positive and negative predictive values were 64%, 91%, 95%, and 49%, respectively. In the falsely positive cases, Alzheimer's disease (n = 4) and vascular dementia (n = 1) were the underlying diseases. However, only in one of these five cases both clinical and EEG data would have led to the false‐positive result to diagnose probable CJD. These data prove the high diagnostic value of our objective EEG criteria in CJD. Ann Neurol 2004
PrPSc, the only known constituent of prions, the infectious agents causing prion diseases, can be detected by real-time quaking-induced conversion (RT-QuIC). However, there is no efficient method to ...quantify the amount of PrPSc by RT-QuIC.
Here we introduce quantitative RT-QuIC (qRT-QuIC) to quantify with high accuracy minute amounts of PrPSc in the brain and various peripheral tissues at levels far below detection by in vivo transmission. PrPSc is relatively resistant to treatment with proteinase K (PK). However, as there can also be a fraction of pathological PrP that is digested by PK, we use the term PrP27-30 to denote to the amount of PrPSc that can be detected by immunoblot after PK treatment. qRT-QuIC is based upon the quantitative correlation between the seeded amount of PrP27-30 and the lag time to the start of the conversion reaction detected by RT-QuIC. By seeding known amounts of PrP27-30 quantified by immunoblot into qRT-QuIC a standard calibration curve can be obtained. Based on this calibration curve, seeded undetermined amounts of PrP27-30 can be directly calculated. qRT-QuIC allowed to quantify PrP27-30 concentrations at extremely low levels as low as 10-15.5 g PrP27-30, which corresponds to 0.001 LD50 units obtained by in vivo i.c. transmission studies. We find that PrP27-30 concentration increases steadily in the brain after inoculation and can be detected at various time points during the incubation period in peripheral organs (spleen, heart, muscle, liver, kidney) in two experimental scrapie strains (RML, ME7) in the mouse.
We suggest that an automatic quantitative system to measure disease progression as well as prion contamination of organs, blood and food product is feasible. Moreover, the concept of qRT-QuIC should be applicable to measure other disease-associated proteins rich in β-pleated structures (amyloid) that bind ThT and that show seeded aggregation.
Oligodendroglial components (OC) and loss of heterozygosity on chromosomes 1p and 19q (LOH 1p/19q) are associated with better outcome in patients with glioma. We aimed to assess the fitness of ...(18)Ffluoroethyltyrosine positron-emission-tomography (FET-PET) for noninvasively identifying these important prognostic/predictive factors. One hundred forty-four patients with MRI-suspected WHO grade II and III glioma underwent FET-PET scans prior to histological diagnosis. FET-PET analyses included maximal tumoral uptake (SUV(max)/BG), biological tumor volume (BTV), mean tumoral uptake (SUV(mean)/BG), total tumoral uptake (SUV(total)/BG), and kinetic analysis. Suspicion of OC was based on static and dynamic FET-uptake parameters. PET results were correlated with histology and 1p/19q status. OC tumors exhibited significantly higher uptake values, compared with astrocytomas (AC) (SUV(max)/BG 3.1 vs 2.3, BTV 15.5 mL vs 7.2 mL, SUV(total)/BG 38.5 vs 17.4, P < .01 each; SUV(mean)/BG 2.2 vs 2.1, P < .05). These differences were more pronounced in WHO grade II gliomas. Comparable results were found with respect to 1p/19q status. Kinetic analysis misclassified 18 of 34 low-grade OC tumors as high-grade glioma but misclassified only 5 of 45 of the low-grade ACs. FET-based suspicion of OC resulted in concordance rates of both 76% for the prediction of OC and LOH 1p/19q. FET-uptake was significantly higher in gliomas with OC, compared with AC, and likewise in 1p/19q codeleted, compared with noncodeleted tumors. However, FET-PET analysis did not reliably predict the presence of OC/LOH 1p/19q in the individual patient, mostly because of an overlap in PET characteristics of OC tumors and high-grade AC. Histological examination is still required for an accurate diagnosis.
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