Thomas Ströbel

A Pan-European Study of the C9orf72 Repeat Associated with FTLD: Geographic Prevalence, Genomic Instability, and Intermediate Repeats

We assessed the geographical distribution of C9orf72 G4C2 expansions in a pan‐European frontotemporal lobar degeneration (FTLD) cohort (n = 1,205), ascertained by the European Early‐Onset Dementia (EOD) consortium. Next, we performed a meta‐analysis of our data and that of other European studies, together 2,668 patients from 15 Western European countries. The frequency of the C9orf72 expansions in Western Europe was 9.98% in overall FTLD, with 18.52% in familial, and 6.26% in sporadic FTLD patients. Outliers were Finland and Sweden with overall frequencies of respectively 29.33% and 20.73%, but also Spain with 25.49%. In contrast, prevalence in Germany was limited to 4.82%. In addition, we studied the role of intermediate repeats (7–24 repeat units), which are strongly correlated with the risk haplotype, on disease and C9orf72 expression. In vitro reporter gene expression studies demonstrated significantly decreased transcriptional activity of C9orf72 with increasing number of normal repeat units, indicating that intermediate repeats might act as predisposing alleles and in favor of the loss‐of‐function disease mechanism. Further, we observed a significantly increased frequency of short indels in the GC‐rich low complexity sequence adjacent to the G4C2 repeat in C9orf72 expansion carriers (P < 0.001) with the most common indel creating one long contiguous imperfect G4C2 repeat, which is likely more prone to replication slippage and pathological expansion.

Brain protein preservation largely depends on the postmortem storage temperature: implications for study of proteins in human neurologic diseases and management of brain banks: a BrainNet Europe Study

The present study was designed to reveal protein modifications in control cases related with postmortem delay and temperature of storage in 3 paradigms in which the same postmortem tissue sample (frontal cortex) was frozen a short time after death or stored at 1°C, 4°C, or room temperature and then frozen at −80°C at different intervals. No evidence of protein degradation as revealed with monodimensional gel electrophoresis and Western blotting was observed in samples artificially stored at 1°C and then frozen at different intervals up to 50 hours after death. However, the levels of several proteins were modified in samples stored at 4°C and this effect was more marked in samples stored at room temperature. Two-dimensional gel electrophoresis and mass spectrometry further corroborated these observations and permitted the identification of other proteins vulnerable or resistant to postmortem delay. Finally, gel electrophoresis and Western blotting of sarkosyl-insoluble fractions in Alzheimer disease showed reduced intensity of phospho-tau-specific bands with postmortem delay with the effects being more dramatic when the brain samples were stored at room temperature for long periods. These results emphasize the necessity of reducing the body temperature after death to minimize protein degradation.

miR-1289 and “Zipcode”-like Sequence Enrich mRNAs in Microvesicles

Despite intensive studies, the molecular mechanisms by which the genetic materials are uploaded into microvesicles (MVs) are still unknown. This is the first study describing a zipcode-like 25 nucleotide (nt) sequence in the 3′-untranslated region (3′UTR) of mRNAs, with variants of this sequence present in many mRNAs enriched in MVs, as compared to their glioblastoma cells of origin. When this sequence was incorporated into the 3′UTR of a reporter message and expressed in a different cell type, it led to enrichment of the reporter mRNA in MVs. Critical features of this sequence are both a CUGCC core presented on a stem-loop structure and a miRNA-binding site, with increased levels of the corresponding miRNA in cells further increasing levels of mRNAs in MVs.

Effects of Formalin Fixation, Paraffin Embedding, and Time of Storage on DNA Preservation in Brain Tissue: A BrainNet Europe Study

There is a large amount of tissue stored in brain collections and brain banks, but little is known about whether formalin‐fixed tissues and paraffin blocks stored for years in brain banks are suitable for the retrospective genetic studies. The study was carried out in order to: (i) compare DNA preservation in frozen, formalin‐fixed and paraffin‐embedded tissues stored for different periods; (ii) study point mutations and triplet expansions in frozen, formalin‐fixed and paraffin‐embedded material stored for variable periods, and using different fixative solutions; (iii) compare different methods to optimize DNA extraction and DNA amplification from suboptimally preserved brain tissue. DNA preservation is suitable for genetic studies in samples stored at −80°C for several years. Formalin‐fixed, paraffin‐embedded tissue was inferior to frozen tissue, but did yield adequate results in many cases depending on the type of fixative solution and time of fixation before embedding. Prolonged fixation in formalin rarely yielded useful DNA. Similar results were obtained in samples from prion diseases. The best results were obtained by using the Qiagen kits (QIAmp DNA Micro) in frozen material, paraffin blocks and formalin‐fixed tissue. Genomiphi™ and TaKaRa Ex Taq™ methods were also assayed in paraffin blocks and in formalin‐fixed samples with limited success.

Genetically Engineered Microvesicles Carrying Suicide mRNA/Protein Inhibit Schwannoma Tumor Growth

Microvesicles (MVs) play an important role in intercellular communication by carrying mRNAs, microRNAs (miRNAs), non-coding RNAs, proteins, and DNA from cell to cell. To our knowledge, this is the first report of delivery of a therapeutic mRNA/protein via MVs for treatment of cancer. We first generated genetically engineered MVs by expressing high levels of the suicide gene mRNA and protein–cytosine deaminase (CD) fused to uracil phosphoribosyltransferase (UPRT) in MV donor cells. MVs were isolated from these cells and used to treat pre-established nerve sheath tumors (schwannomas) in an orthotopic mouse model. We demonstrated that MV-mediated delivery of CD-UPRT mRNA/protein by direct injection into schwannomas led to regression of these tumors upon systemic treatment with the prodrug (5-fluorocytosine (5-FC)), which is converted within tumor cells to 5-fluorouracil (5-FU)–an anticancer agent. Taken together, these studies suggest that MVs can serve as novel cell-derived “liposomes” to effectively deliver therapeutic mRNA/proteins to treatment of diseases.

Genetic Creutzfeldt-Jakob disease associated with the E200K mutation: characterization of a complex proteinopathy

The E200K mutation is the most frequent prion protein gene (PRNP) mutation detected worldwide that is associated with Creutzfeldt-Jakob disease (CJD) and thought to have overlapping features with sporadic CJD, yet detailed neuropathological studies have not been reported. In addition to the prion protein, deposition of tau, α-synuclein, and amyloid-β has been reported in human prion disease. To describe the salient and concomitant neuropathological alterations, we performed a systematic clinical, neuropathological, and biochemical study of 39 individuals carrying the E200K PRNP mutation originating from different European countries. The most frequent clinical symptoms were dementia and ataxia followed by myoclonus and various combinations of further symptoms, including vertical gaze palsy and polyneuropathy. Neuropathological examination revealed relatively uniform anatomical pattern of tissue lesioning, predominating in the basal ganglia and thalamus, and also substantia nigra, while the deposition of disease-associated PrP was more influenced by the codon 129 constellation, including different or mixed types of PrPres detected by immunoblotting. Unique and prominent intraneuronal PrP deposition involving brainstem nuclei was also noted. Systematic examination of protein depositions revealed parenchymal amyloid-β in 53.8%, amyloid angiopathy (Aβ) in 23.1%, phospho-tau immunoreactive neuritic profiles in 92.3%, neurofibrillary degeneration in 38.4%, new types of tau pathology in 33.3%, and Lewy-type α-synuclein pathology in 15.4%. TDP-43 and FUS immunoreactive protein deposits were not observed. This is the first demonstration of intensified and combined neurodegeneration in a genetic prion disease due to a single point mutation, which might become an important model to decipher the molecular interplay between neurodegeneration-associated proteins.

Involvement of the endosomal-lysosomal system correlates with regional pathology in creutzfeldt-jakob disease

The endosomal-lysosomal system (ELS) has been suggested to play a role in the pathogenesis of prion diseases. The purpose of this study was to examine how experimental observations can be translated to human neuropathology and whether alterations of the ELS relate to neuropathologic changes. Combined with stereologic techniques, we examined components of the ELS in human sporadic Creutzfeldt-Jakob disease brains. We immunostained for the early endosomal marker Rab5 and lysosomal enzymes cathepsin D and B. We determined neuron-specific changes in their expression and correlated these with the severity of neuropathologic changes. In regions with mild pathology and scant abnormal prion protein (PrPSc) deposition, neurons showed an increased volume of Rab5-immunopositive early endosomes. In contrast, neurons in regions with prominent pathology had an increased volume of cathepsin D- or B-immunoreactive lysosomes. The intraneuronal distribution of cathepsin D and B diverges between Purkinje cells and frontal cortical neurons in sporadic Creutzfeldt-Jakob disease brains. We demonstrated focal intra- and perineuronal colocalization of cathepsin D and PrPSc. Our results indicate that effects in the ELS correlate with regional pathology. Overloading of this system might impair the function of lysosomal enzymes and thus may mimic some features of lysosomal storage disorders.

Creutzfeldt–Jakob disease and inclusion body myositis: Abundant disease‐associated prion protein in muscle

Pathologicalprion protein (PrPSc) is the hallmark of prion diseases affecting primarily the central nervous system. Using immunohistochemistry, paraffin‐embedded tissue blot, and Western blot, we demonstrated abundant PrPSc in the muscle of a patient with sporadic Creutzfeldt–Jakob disease and inclusion body myositis. Extraneural PrPC‐PrPSc conversion in Creutzfeldt–Jakob disease appears to become prominent when PrPC is abundantly available as substrate, as in inclusion body myositis muscle.

Interlaboratory comparison of IDH mutation detection

Isocitrate dehydrogenase (IDH) mutational testing is becoming increasingly important. For this, robust and reliable assays are needed. We tested the variation of results between six laboratories of testing for IDH mutations. Each laboratory received five unstained slides from 31 formalin-fixed paraffin-embedded (FFPE) glioma samples, and followed its own standard IDH diagnostic routine. All laboratories used immunohistochemistry (IHC) with an antibody against the most frequent IDH1 mutation (R132H) as a first step. Three laboratories then sequenced only IHC negative cases while the others sequenced all cases. Based on the overall analysis, 13 samples from 11 tumors had an R132H mutation and one tumor showed an R132G mutation. Results of IHC for IDH1 R132H mutations in all six laboratories were completely in agreement, and identified all R132H mutations. Upon sequencing the results of two laboratories deviated from those of the others. After a review of the entire diagnostic process, on repeat (blinded) testing one laboratory was completely in agreement with the overall result. A change in technique did only partially improve the results in the other laboratory. IHC for the IDH1 R132H mutation is very reliable and consistent across laboratories. IDH sequencing procedures yielded inconsistent results in 2 out of 6 laboratories. Quality assurance is pivotal before IDH testing is made part of clinical management of patients.

Inter-laboratory assessment of PrPSc typing in Creutzfeldt-Jakob disease: a western blot study within the NeuroPrion consortium.

Molecular typing is of considerable importance for the surveillance and epidemiology of human transmissible spongiform encephalopathies (TSEs). It relies on the detection of distinct protease‐resistant prion protein (PrPSc) core fragments that differ in molecular mass and/or glycoform ratio. In this collaborative study, we tested the inter‐laboratory agreement in TSE molecular typing. Sixteen characterized brain specimens from sporadic TSEs and variant Creutzfeldt‐Jakob disease (vCJD) cases were distributed blindly to seven laboratories for molecular characterization by a defined protocol and classification. Agreement between laboratories in the classification of samples was excellent. In particular, there were no differences in the distinction between PrPSc type 1, type 2A, and type 2B with one exception, which eventually was identified as a case with types 1 and 2 co‐occurrence. This shows that the general technique and particular classification system used here are robust and represent a reliable basis for diagnostic and epidemiologic purposes. The subtle further distinction of subtypes among type 1 and type 2 groups requires high‐sensitivity gel electrophoresis protocols that are unsuitable for routine diagnostic needs and must be reserved for research investigations. Further research is necessary on the identification and significance of co‐occurrence of PrPSc types 1 and 2 within one brain.