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-β 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 × 103 M− 1 s− 1. Fibril elongation of α-syn is slow compared to other amyloidogenic proteins.

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.

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.

Recent studies indicate that small amyloid-β peptide (Aβ) oligomers are the major toxic species responsible for development and progression of Alzheimer's disease (AD). Therefore, we suggest that the number of Aβ oligomers in body fluids is the most direct and relevant biomarker for AD. Determination of the Aβ 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 Aβ oligomers, while the samples of the AD group exhibited significantly higher levels of Aβ oligomers. The Aβ oligomer numbers correlated with the patients' Mini-Mental State Examination scores. This indicates that the quantity of Aβ oligomers in CSF reflects the severity of the disease and that Aβ oligomers play a crucial role in AD pathology and in turn can be used as a diagnostic biomarker.

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 (PrP C ) into its misfolded pathogenic isoform (PrP Sc ). PrP Sc is the main component of the pathological and infectious prion agent. The study of the conversion mechanism from PrP C to PrP Sc is a major field in prion research. PrP C 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 PrP C in the yeast Pichia pastoris using native PrP C -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 PrP C , resulting in PrP C localised in the plasma membrane of P. pastoris . In addition, the glycosylation pattern of native PrP C could be confirmed.

Alzheimer's disease (AD) is a fatal neurodegenerative and progressive disorder. Currently, no reliable biomarker for pre-symptomatic diagnosis or therapy monitoring is available. Recent studies support that especially soluble Aβ oligomers are the major toxic species during development and progression of AD. Therefore, we suggest that the number of Aβ oligomers in body fluids can be used as the most direct biomarker for AD. Here, we present an optimized version of the single aggregate sensitive surface-based fluorescence intensity distribution analysis (sFIDA) assay. For the first time, it allowed the determination of the Aβ oligomer count in cerebrospinal fluid (CSF). We challenged the assay with CSF samples of 14 AD patients and 12 age-matched control subjects. The Aβ oligomer count allowed a surprisingly clear distinction between both groups. All samples of the control group showed homogenously low numbers of Aβ oligomers. The samples of the AD group had comparably high levels of Aβ oligomers and displayed high variability. The Aβ oligomer levels clearly correlated with the patients' mini-mental state examination (MMSE) scores. Our results support the idea that Aβ oligomers play a decisive role in AD pathology and in turn can be used as a diagnostic biomarker. The sFIDA assay is able to reliably quantify the Aβ oligomers in human CSF. The correlation between MMSE and Aβ oligomer count suggests that the quantitity of Aβ oligomers in CSF even reflects the severity of the disease. Further studies will show whether anti-Aβ targeted therapies can affect the Aβ oligomer counts in treated individuals.

altered blood thiamine metabolism was associated with reduced activities of thiamine pyrophosphokinase in patients with Alzheiemr’s disease as compared with control subjects (1.24 6 0.06 vs. 1.44 6 0.07, P<0.05). Conclusions: The patients with Alzheimer’s disease manifest significant abnormality in blood thiamine metabolism as compared with normal cognitive subjects and patients with vascular dementia. Altered blood thiamine metabolism represents an ideal diagnostic biomarker for Alzheimer’s disease with the reliable, non-invasive, simple to perform and inexpensive merits, and is associated with decreased thiamine pyrophosphokinase activities.

Parkinson disease (PD) is one of the most common age-related neurodegenerative diseases associated with motor deficiencies in humans. The symptoms are caused by the death of dopaminergic neurons in the brain, which is accompanied by the misfolding and aggregation of the protein α-synuclein. Diagnosis is based on the incidence of clinical symptoms, although they only appear as a result of the irreversible damage of neurons during the disease. Identification of a suitable biomarker would allow preclinical diagnosis. We an approach to quantify single α-synuclein aggregates as a possible biomarker for PD.

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.