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 Parkinson's disease (PD) or amyloid-ß in Alzheimer's 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.

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 (PrP(C)) to its pathological isoform PrP(Sc), 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 PrP(Sc) 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 PrP(C) and PrP(Sc). 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 PrP(Sc) as seed is sufficient to explain the phenomenon of species barriers. Therefore our data supports the hypothesis that CWD is not transmissible to humans.

Expression and characterisation of fully posttranslationally modified cellular prion protein in Pichia pastoris.

Prion diseases are fatal neurodegenerative diseases which occur as sporadic, genetic, and transmissible disorders. A molecular hallmark of prion diseases is the conformational conversion of the host-encoded cellular form of the prion protein (PrPC) into its misfolded pathogenic isoform (PrPSc). PrPSc is the main component of the pathological and infectious prion agent. The study of the conversion mechanism from PrPC to PrPSc is a major field in prion research. PrPC is glycosylated and attached to the plasma membrane via its glycosyl phosphatidyl inositol (GPI)-anchor. In this study we established and characterised the expression of fully posttranslationally modified mammalian Syrian golden hamster PrPC in the yeast Pichia pastoris using native PrPC-specific N- and C-terminal signal sequences. In vivo as well as in vitro-studies demonstrated that the signal sequences controlled posttranslational processing and trafficking of native PrPC, resulting in PrPC localised in the plasma membrane of P. pastoris. In addition, the glycosylation pattern of native PrPC could be confirmed.

The amyloid-ß oligomer count in cerebrospinal fluid is a biomarker for Alzheimer's disease.

Recent studies indicate that small amyloid-ß peptide (Aß) oligomers are the major toxic species responsible for development and progression of Alzheimer's disease (AD). Therefore, we suggest that the number of Aß oligomers in body fluids is the most direct and relevant biomarker for AD. Determination of the Aß oligomer content of cerebrospinal fluid (CSF) samples from 14 AD patients and 12 age-matched controls revealed a clear distinction between both groups. All samples of the control group showed homogenously low numbers of Aß oligomers, while the samples of the AD group exhibited significantly higher levels of Aß oligomers. The Aß oligomer numbers correlated with the patients' Mini-Mental State Examination scores. This indicates that the quantity of Aß oligomers in CSF reflects the severity of the disease and that Aß oligomers play a crucial role in AD pathology and in turn can be used as a diagnostic biomarker.

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.

Kinetics of advanced glycation end products formation on bovine serum albumin with various reducing sugars and dicarbonyl compounds in equimolar ratios.

Reducing sugars and reactive dicarbonyl compounds play a major role in glycation of proteins in vivo. Glycation of proteins is the first step in of a nonenzymatic reaction, resulting in advanced glycation end products (AGEs). AGEs can inactivate proteins or modify their biological activities. Therefore, it is important to understand the mechanism of AGE formation. Here, we systematically analyzed the kinetics of AGE formation in vitro by fluorescence and absorption measurements utilizing a microplate reader system and bovine serum albumin (BSA) as a model protein. Comparing different concentrations of BSA, we applied various reducing sugars and reactive dicarbonyl compounds as AGE-inducing agents at different concentrations. In summary, this experimental setup enabled us to measure the kinetics of AGE formation in an efficient and defined way.

In vitro conversion and seeded fibrillization of posttranslationally modified prion protein.

The conversion of the cellular isoform of the prion protein (PrP(C)) into the pathologic isoform (PrP(Sc)) is the key event in prion diseases. To study the conversion process, an in vitro system based on varying the concentration of low amounts of sodium dodecyl sulfate (SDS) has been employed. In the present study, the conversion of full-length PrP(C) isolated from Chinese hamster ovary cells (CHO-PrP(C)) was examined. CHO-PrP(C) harbors native, posttranslational modifications, including the GPI anchor and two N-linked glyco-sylation sites. The properties of CHO-PrP(C) were compared with those of full-length and N-terminally truncated recombinant PrP. As shown earlier with recombinant PrP (recPrP90-231), transition from a soluble α-helical state as known for native PrP(C) into an aggregated, ß-sheet-rich PrP(Sc)-like state could be induced by dilution of SDS. The aggregated state is partially proteinase K (PK)-resistant, exhibiting a cleavage site similar to that found with PrP(Sc). Compared to recPrP (90-231), fibril formation with CHO-PrP(C) requires lower SDS concentrations (0.0075%), and can be drastically accelerated by seeding with PrP(Sc) purified from brain homogenates of terminally sick hamsters. Our results show that recPrP 90-231 and CHO-PrPC behave qualitatively similar but quantitatively different. The in vivo situation can be simulated closer with CHO-PrP(C) because the specific PK cleave site could be shown and the seed-assisted fibrillization was much more efficient.

Molecular interactions between prions as seeds and recombinant prion proteins as substrates resemble the biological interspecies barrier in vitro.

Prion diseases like Creutzfeldt-Jakob disease in humans, Scrapie in sheep or bovine spongiform encephalopathy are fatal neurodegenerative diseases, which can be of sporadic, genetic, or infectious origin. Prion diseases are transmissible between different species, however, with a variable species barrier. The key event of prion amplification is the conversion of the cellular isoform of the prion protein (PrP(C)) into the pathogenic isoform (PrP(Sc)). We developed a sodiumdodecylsulfate-based PrP conversion system that induces amyloid fibril formation from soluble α-helical structured recombinant PrP (recPrP). This approach was extended applying pre-purified PrP(Sc) as seeds which accelerate fibrillization of recPrP. In the present study we investigated the interspecies coherence of prion disease. Therefore we used PrP(Sc) from different species like Syrian hamster, cattle, mouse and sheep and seeded fibrillization of recPrP from the same or other species to mimic in vitro the natural species barrier. We could show that the in vitro system of seeded fibrillization is in accordance with what is known from the naturally occurring species barriers.

Influence of the maillard reaction to prion protein aggregation.

Prion diseases are fatal neurodegenerative diseases that occur either spontaneously or genetically or are caused by infection. Spontaneously occurring prion diseases are age related. The infectious agents, called prions, are proteinaceous infectious particles, composed mainly of the host-encoded prion protein (PrP) in a misfolded, insoluble, and aggregated isoform. Advanced glycation end products (AGEs) are well known to contribute to protein misfolding, insolubility, and aggregation. Thus, we studied if AGE-modification could influence PrP aggregation. We analyzed PrP preparations immunochemically to determine if they contain AGE-modified PrP. We also studied the influence of AGE modifications on the PrP aggregation process in vitro.

Single-particle detection system for Abeta aggregates: adaptation of surface-fluorescence intensity distribution analysis to laser scanning microscopy.

Today, Alzheimer disease (AD) can be diagnosed with certainty only post mortem. A biomarker method for early diagnosis of AD is urgently needed. Abeta aggregates are directly involved in AD progression and therefore might be useful as biomarker in body fluids. We have developed an ultrasensitive assay system for the detection of Abeta aggregates in body fluids, called surface-fluorescence intensity distribution analysis (FIDA). Surface-FIDA in its first version was based on fluorescence correlation spectroscopy. Here we show that surface-FIDA can also be performed using a laser scanning microscope setup and is then nicely suitable for the characterization of Abeta aggregates.

Prion infection: seeded fibrillization or more?

The prion infection is a conversion of host encoded prion protein (PrP) from its cellular isoform PrP(C) into the pathological and infectious isoform PrP(Sc); the conversion process was investigated by in vitro studies using recombinant and cellular PrP and natural PrP(Sc). We present a brief summary of the results determined with our in vitro conversion system and the derived mechanistic models. We describe well characterized intermediates and precursor states during the conversion process, kinetic studies of spontaneous and seeded fibrillogenesis and the impact of the membrane environment.

Spontaneous and BSE-prion-seeded amyloid formation of full length recombinant bovine prion protein.

The conversion of the cellular isoform of the prion protein into the pathogenic isoform PrP(Sc) is the key event in prion diseases. The disease can occur spontaneously genetically or by infection. In earlier studies we presented an in vitro conversion system which simulates the structural transition in recPrP by varying low concentrations of SDS at constant NaCl. In the present study we adopted the conversion system from experimental Scrapie in hamster to bovine recPrP and generated amyloid fibrils. The intermediate state which is optimal for fibril formation is a soluble, beta-rich state. The system was extended using BSE-prions as seeds and led to an acceleration of fibril formation by orders of magnitude. This seeded amyloid formation assay avoids any PK-treatment, is therefore able to detect even PK-sensitive PrP(Sc) and does not require cellular components.

A highly sensitive diagnostic assay for aggregate-related diseases, including prion diseases and Alzheimer's disease.

Prion diseases, Alzheimer's disease, and Parkinson's disease are age-related neurodegenerative diseases that are characterized by the formation of protein aggregates during the progress of the disease. Although it is still not known whether these aggregates are causative for, or symptoms of, the disease. Many studies show that aggregates or even oligomers of the according proteins are neurotoxic and thus may lead to neurodegeneration. To understand disease-associated or causative mechanisms in respect to protein aggregation, an ultrasensitive tool to quantify these disease-related aggregates is required. In this study we introduce a specificity-enhanced version of surface-FIDA as an approach to count even single aggregates in tissue homogenate and body liquids.

Aggregation and amyloid fibril formation of the prion protein is accelerated in the presence of glycogen.

Prion diseases like Creutzfeldt-Jakob disease in humans or scrapie in sheep and goats are infectious neurodegenerative diseases. Their infectious agent, called prion, is composed mainly of aggregated and misfolded prion protein and non-proteinaceous components. An example of such a common non-proteinaceous secondary component of natural prions is the polysaccharide scaffold. We studied the influence of such a polysaccharide on the conformational transition of PrP applying an in vitro conversion system. Here we report that glycogen supports and accelerates PrP amorphous aggregation similar to seeded aggregation and leads to co-aggregates. Furthermore, PrP fibril formation was highly accelerated in the presence of glycogen.

An ultrasensitive assay for diagnosis of Alzheimer's disease.

Alzheimer's disease (AD) is a chronic neurodegenerative disorder and the most common cause of dementia. Aging is among the most significant risk factors. Today, AD can be diagnosed with certainty only post mortem, detecting insoluble beta-amyloid peptide (Abeta) aggregates in the patient's brain tissue. We have developed an ultrasensitive assay for early and non-invasive diagnosis of AD. This highly specific and sensitive assay uses fluorescence correlation spectroscopy (FCS) and is sensitive enough to detect even single aggregates in body fluids of AD patients. We investigate the correlation of aggregated Abeta concentrations in body fluids with clinical symptoms of AD.

Mechanisms of prion protein assembly into amyloid.

The conversion of the alpha-helical, cellular isoform of the prion protein (PrP(C)) to the insoluble, beta-sheet-rich, infectious, disease-causing isoform (PrP(Sc)) is the key event in prion diseases. In an earlier study, several forms of PrP were converted into a fibrillar state by using an in vitro conversion system consisting of low concentrations of SDS and 250 mM NaCl. Here, we characterize the structure of the fibril precursor state, that is, the soluble state under fibrillization conditions. CD spectroscopy, analytical ultracentrifugation, and chemical cross-linking indicate that the precursor state exists in a monomer-dimer equilibrium of partially denatured, alpha-helical PrP, with a well defined contact site of the subunits in the dimer. Using fluorescence with thioflavin T, we monitored and quantitatively described the kinetics of seeded fibril formation, including dependence of the reaction on substrate and seed concentrations. Exponential, seed-enhanced growth can be achieved in homogeneous solution, which can be enhanced by sonication. From these data, we propose a mechanistic model of fibrillization, including the presence of several intermediate structures. These studies also provide a simplified amplification system for prions.

Single particle detection of Abeta aggregates associated with Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder and the most common cause of dementia. Today, AD can be diagnosed with certainty only post-mortem, by histopathologic staining of Abeta plaques and neurofibrillary tangles in brain tissue sections. We have developed an ultra-sensitive assay potentially suitable for early and non-invasive diagnosis of AD. This highly specific and sensitive assay uses fluorescence correlation spectroscopy (FCS) and is sensitive enough to detect even single aggregates in body fluids of AD patients. First results show a clear distinction between AD diseased people and non-demented controls by analysing cerebrospinal fluids (CSF) by confocal scanning of surface captured Abeta aggregates and subsequent two-dimensional fluorescence intensity distribution analysis.

Counting of single prion particles bound to a capture-antibody surface (surface-FIDA).

Hitherto accredited prion tests use the PK resistance of PrP(Sc), the pathogenic isoform of the prion protein, as a marker for the disease. Because of variations in the amount of disease-related aggregated PrP, which is not PK-resistant, these prion tests offer only limited sensitivity. Therefore, a prion detection method that does not rely on PK digestion would allow for the detection of both PK-resistant as well as PK-sensitive PrP(Sc). Furthermore, single particle counting is more sensitive than methods measuring an integrated signal. Our new test system is based on dual-colour fluorescence correlation spectroscopy (FCS). This method quantifies the number of protein aggregates that have been simultaneously labelled with two different antibodies using dual-colour fluorescence intensity distribution analysis (2D-FIDA). This only counts PrP aggregates, and not PrP monomers. To increase the sensitivity, PrP(Sc) was concentrated in a two-dimensional space by immobilizing it so that the antibodies could be captured on the surface of the slide (surface-FIDA). When the surface was systematically scanned, even single prion particles were detected. Using this new technique, the sensitivity to identify samples from scrapie-infected hamster as well as BSE-infected cattle can be dramatically increased in comparison with identification using FIDA in solution.

Detection of prion particles in samples of BSE and scrapie by fluorescence correlation spectroscopy without proteinase K digestion.

A characteristic feature of prion diseases such as bovine spongiform encephalopathy (BSE) is the accumulation of a pathological isoform of the host-encoded prion protein, PrP. In contrast to its cellular isoform PrP(C), the pathological isoform PrP(Sc) forms insoluble aggregates. All commercial BSE tests currently used for routine testing are based on the proteinase K (PK) resistance of PrP, but not all pathological PrP is PK-resistant. In the present study, single prion particles were counted by fluorescence correlation spectroscopy (FCS). The property of PK resistance is not required, i.e., both the PK-resistant and the PK-sensitive parts of the prion particles are detectable. PrP aggregates were prepared from the brains of BSE-infected cattle, as well as from scrapie-infected hamsters, by the NaPTA precipitation method without PK digestion. They were labeled using two different PrP-specific antibodies for FCS measurements in the dual-color mode (2D-FIDA). Within the limited number of samples tested, BSE-infected cattle and scrapie-infected hamsters in the clinical stage of the disease could be distinguished with 100% specificity from a control group. Thus, a diagnostic tool for BSE detection with complete avoidance of PK treatment is presented, which should have particular advantages for testing animals in the preclinical stage.