Oliver Bannach

Accumulation of viroid-specific small RNAs and increase in nucleolytic activities linked to viroid-caused pathogenesis.

Abstract Strong viroid-caused pathogenesis was achieved in tomato cv. Rutgers by biolistic transfer of severe or lethal potato spindle tuber viroid (PSTVd) strains, while other tomato genotypes (e.g., Moneymaker) were tolerant. With reciprocal hybrids between sensitive and tolerant genotypes, we show that plant depression dominates over tolerance. Biolistic transfer of the most pathogenic PSTVd strain AS1 to Nicotiana benthamiana, which is considered to be a symptomless PSTVd host, led to a strong pathogenesis reaction and stunting, suggesting the presence of specific viroid pathogenesis-promoting target(s) in this plant species. Total levels of small siRNA-like PSTVd-specific RNAs were enhanced in strongly symptomatic tomato and N. benthamiana plants after biolistic infection with AS1 in comparison to the mild QFA strain. This indicates association of elevated levels of viroid-specific small RNA with production of strong symptoms. In symptom-bearing tomato leaves in comparison to controls, an RNase of approximately 18 kDa was induced and the activity of a nuclease of 34 kDa was elevated by a factor of seven in the vascular system. Sequence analysis of the nuclease cDNA designated TBN1 showed high homology with plant apoptotic endonucleases. The vascular-specific pathogenesis action is supported by light microscopic observations demonstrating a certain lack of xylem tissue and an arrest of the establishment of new vascular bundles in collapsed plants.

Detection of prion protein particles in blood plasma of scrapie infected sheep.

Prion diseases are transmissible neurodegenerative diseases affecting humans and animals. The agent of the disease is the prion consisting mainly, if not solely, of a misfolded and aggregated isoform of the host-encoded prion protein (PrP). Transmission of prions can occur naturally but also accidentally, e.g. by blood transfusion, which has raised serious concerns about blood product safety and emphasized the need for a reliable diagnostic test. In this report we present a method based on surface-FIDA (fluorescence intensity distribution analysis), that exploits the high state of molecular aggregation of PrP as an unequivocal diagnostic marker of the disease, and show that it can detect infection in blood. To prepare PrP aggregates from blood plasma we introduced a detergent and lipase treatment to separate PrP from blood lipophilic components. Prion protein aggregates were subsequently precipitated by phosphotungstic acid, immobilized on a glass surface by covalently bound capture antibodies, and finally labeled with fluorescent antibody probes. Individual PrP aggregates were visualized by laser scanning microscopy where signal intensity was proportional to aggregate size. After signal processing to remove the background from low fluorescence particles, fluorescence intensities of all remaining PrP particles were summed. We detected PrP aggregates in plasma samples from six out of ten scrapie-positive sheep with no false positives from uninfected sheep. Applying simultaneous intensity and size discrimination, ten out of ten samples from scrapie sheep could be differentiated from uninfected sheep. The implications for ante mortem diagnosis of prion diseases are discussed.

Alternative splicing of anciently exonized 5S rRNA regulates plant transcription factor TFIIIA

Identifying conserved alternative splicing (AS) events among evolutionarily distant species can prioritize AS events for functional characterization and help uncover relevant cis- and trans-regulatory factors. A genome-wide search for conserved cassette exon AS events in higher plants revealed the exonization of 5S ribosomal RNA (5S rRNA) within the gene of its own transcription regulator, TFIIIA (transcription factor for polymerase III A). The 5S rRNA-derived exon in TFIIIA gene exists in all representative land plant species but not in green algae and nonplant species, suggesting it is specific to land plants. TFIIIA is essential for RNA polymerase III-based transcription of 5S rRNA in eukaryotes. Integrating comparative genomics and molecular biology revealed that the conserved cassette exon derived from 5S rRNA is coupled with nonsense-mediated mRNA decay. Utilizing multiple independent Arabidopsis overexpressing TFIIIA transgenic lines under osmotic and salt stress, strong accordance between phenotypic and molecular evidence reveals the biological relevance of AS of the exonized 5S rRNA in quantitative autoregulation of TFIIIA homeostasis. Most significantly, this study provides the first evidence of ancient exaptation of 5S rRNA in plants, suggesting a novel gene regulation model mediated by the AS of an anciently exonized noncoding element.

Single Fibril Growth Kinetics of α-Synuclein

Neurodegenerative disorders associated with protein misfolding are fatal diseases that are caused by fibrillation of endogenous proteins such as α-synuclein (α-syn) in Parkinsons disease (PD) or amyloid-β in Alzheimers disease. Fibrils of α-syn are a major pathological hallmark of PD and certain aggregation intermediates are postulated to cause synaptic failure and cell death of dopaminergic neurons in the substantia nigra. For the development of therapeutic approaches, the mechanistic understanding of the fibrillation process is essential. Here we report real-time observation of α-syn fibril elongation on a glass surface, imaged by total internal reflection fluorescence microscopy using thioflavin T fluorescence. Fibrillation on the glass surface occurred in the same time frame and yielded fibrils of similar length as fibrillation in solution. Time-resolved imaging of fibrillation on a single fibril level indicated that α-syn fibril elongation follows a stop-and-go mechanism; that is, fibrils either extend at a homogenous growth rate or stop to grow for variable time intervals. The fibril growth kinetics were compatible with a model featuring two states, a growth state and a stop state, which were approximately isoenergetic and interconverted with rate constants of ~1.5×10(-4) s(-1). In the growth state, α-syn monomers were incorporated into the fibril with a rate constant of 8.6×10(3) M(-1) s(-1). Fibril elongation of α-syn is slow compared to other amyloidogenic proteins.

Application of an Amyloid Beta Oligomer Standard in the sFIDA Assay

Still, there is need for significant improvements in reliable and accurate diagnosis for Alzheimers disease (AD) at early stages. It is widely accepted that changes in the concentration and conformation of amyloid-β (Aβ) appear several years before the onset of first symptoms of cognitive impairment in AD patients. Because Aβ oligomers are possibly the major toxic species in AD, they are a promising biomarker candidate for the early diagnosis of the disease. To date, a variety of oligomer-specific assays have been developed, many of them ELISAs. Here, we demonstrate the sFIDA assay, a technology highly specific for Aβ oligomers developed toward single particle sensitivity. By spiking stabilized Aβ oligomers to buffer and to body fluids from control donors, we show that the sFIDA readout correlates with the applied concentration of stabilized oligomers diluted in buffer, cerebrospinal fluid (CSF), and blood plasma over several orders of magnitude. The lower limit of detection was calculated to be 22 fM of stabilized oligomers diluted in PBS, 18 fM in CSF, and 14 fM in blood plasma.

Seeded Fibrillation as Molecular Basis of the Species Barrier in Human Prion Diseases

Prion diseases are transmissible spongiform encephalopathies in humans and animals, including scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease (CWD) in deer, and Creutzfeldt-Jakob disease (CJD) in humans. The hallmark of prion diseases is the conversion of the host-encoded prion protein (PrPC) to its pathological isoform PrPSc, which is accompanied by PrP fibrillation. Transmission is not restricted within one species, but can also occur between species. In some cases a species barrier can be observed that results in limited or unsuccessful transmission. The mechanism behind interspecies transmissibility or species barriers is not completely understood. To analyse this process at a molecular level, we previously established an in vitro fibrillation assay, in which recombinant PrP (recPrP) as substrate can be specifically seeded by PrPSc as seed. Seeding with purified components, with no additional cellular components, is a direct consequence of the “prion-protein-only” hypothesis. We therefore hypothesise, that the species barrier is based on the interaction of PrPC and PrPSc. Whereas in our earlier studies, the interspecies transmission in animal systems was analysed, the focus of this study lies on the transmission from animals to humans. We therefore combined seeds from species cattle, sheep and deer (BSE, scrapie, CWD) with human recPrP. Homologous seeding served as a control. Our results are consistent with epidemiology, other in vitro aggregation studies, and bioassays investigating the transmission between humans, cattle, sheep, and deer. In contrast to CJD and BSE seeds, which show a seeding activity we can demonstrate a species barrier for seeds from scrapie and CWD in vitro. We could show that the seeding activity and therewith the molecular interaction of PrP as substrate and PrPSc as seed is sufficient to explain the phenomenon of species barriers. Therefore our data supports the hypothesis that CWD is not transmissible to humans.

Contact between the β1 and β2 Segments of α-Synuclein that Inhibits Amyloid Formation

Conversion of the intrinsically disordered protein α-synuclein (α-syn) into amyloid aggregates is a key process in Parkinsons disease. The sequence region 35-59 contains β-strand segments β1 and β2 of α-syn amyloid fibril models and most disease-related mutations. β1 and β2 frequently engage in transient interactions in monomeric α-syn. The consequences of β1-β2 contacts are evaluated by disulfide engineering, biophysical techniques, and cell viability assays. The double-cysteine mutant α-synCC, with a disulfide linking β1 and β2, is aggregation-incompetent and inhibits aggregation and toxicity of wild-type α-syn. We show that α-syn delays the aggregation of amyloid-β peptide and islet amyloid polypeptide involved in Alzheimers disease and type 2 diabetes, an effect enhanced in the α-synCC mutant. Tertiary interactions in the β1-β2 region of α-syn interfere with the nucleation of amyloid formation, suggesting promotion of such interactions as a potential therapeutic approach.

Biofunctionalized Silica Nanoparticles: Standards in Amyloid-β Oligomer-Based Diagnosis of Alzheimer’s Disease

Amyloid-β (Aβ) oligomers represent a promising biomarker for the early diagnosis of Alzheimers disease (AD). However, state-of-the-art methods for immunodetection of Aβ oligomers in body fluids show a large variability and lack a reliable and stable standard that enables the reproducible quantitation of Aβ oligomers. At present, the only available standard applied in these assays is based on a random aggregation process of synthetic Aβ and has neither a defined size nor a known number of epitopes. In this report, we generated a highly stable standard in the size range of native Aβ oligomers that exposes a defined number of epitopes. The standard consists of a silica nanoparticle (SiNaP), which is functionalized with Aβ peptides on its surface (Aβ-SiNaP). The different steps of Aβ-SiNaP synthesis were followed by microscopic, spectroscopic and biochemical analyses. To investigate the performance of Aβ-SiNaPs as an appropriate standard in Aβ oligomer immunodetection, Aβ-SiNaPs were diluted in cerebrospinal fluid and quantified down to a concentration of 10 fM in the sFIDA (surface-based fluorescence intensity distribution analysis) assay. This detection limit corresponds to an Aβ concentration of 1.9 ng l-1 and lies in the sensitivity range of currently applied diagnostic tools based on Aβ oligomer quantitation. Thus, we developed a highly stable and well-characterized standard for the application in Aβ oligomer immunodetection assays that finally allows the reproducible quantitation of Aβ oligomers down to single molecule level and provides a fundamental improvement for the worldwide standardization process of diagnostic methods in AD research.

Analysis of prion protein aggregates in blood and brain from pre-clinical and clinical BSE cases

Prion diseases are infectious neurodegenerative diseases affecting humans and animals. The food-borne bovine spongiform encephalopathy (BSE) had serious impact on both economy and public health, respectively. To follow the pathogenesis of BSE, oral challenge studies were previously conducted, among others on the Isle of Riems, Germany (Balkema-Buschmann et al., 2011b). In the present work brain and plasma samples from this pathogenesis study were subjected to surface fluorescence distribution analysis (sFIDA). sFIDA is a diagnostic tool that exploits the aggregated state of the disease-related prion protein (PrP) as a biomarker for prion disorders. With the exception of one animal, all tested brain samples from clinical cattle exhibited a high titer of PrP particles. Moreover we could detect PrP aggregates already 16 and 24 months after infection. In contrast to our previous demonstration of PrP particles in blood plasma from scrapie sheep, however, no aggregates could be identified in plasma from pre-clinical and clinical cattle. This is in accordance with other studies suggesting a restriction of the BSE infection to the central nervous system.

Analysis of anticoagulants for blood-based quantitation of amyloid β oligomers in the sFIDA assay

Abstract Early diagnostics at the preclinical stage of Alzheimer’s disease is of utmost importance for drug development in clinical trials and prognostic guidance. Since soluble Aβ oligomers are considered to play a crucial role in the disease pathogenesis, several methods aim to quantify Aβ oligomers in body fluids such as cerebrospinal fluid (CSF) and blood plasma. The highly specific and sensitive method surface-based fluorescence intensity distribution analysis (sFIDA) has successfully been established for oligomer quantitation in CSF samples. In our study, we explored the sFIDA method for quantitative measurements of synthetic Aβ particles in blood plasma. For this purpose, EDTA-, citrate- and heparin-treated blood plasma samples from five individual donors were spiked with Aβ coated silica nanoparticles (Aβ-SiNaPs) and were applied to the sFIDA assay. Based on the assay parameters linearity, coefficient of variation and limit of detection, we found that EDTA plasma yields the most suitable parameter values for quantitation of Aβ oligomers in sFIDA assay with a limit of detection of 16 fM.