Amyloid Amplification Assays for the Sensitive Detection of Protein Aggregates

Abstract

Protein misfolding and aggregation diseases such as Alzheimer’s, Parkinson’s or prion diseases are devastating neurodegenerative disorders that strongly affect the ageing population. Prolonged life expectancy and the fact that age represents a crucial risk factor for these disorders will exacerbate the problem in the future. As a consequence, there is an urgent need for diagnostic tests that allow early and reliable diagnosis of these diseases. The main pathological hallmark of protein misfolding and aggregation diseases is the accumulation of proteins as amyloid aggregates. Recently developed biochemical assays aim to amplify and detect small amounts of protein aggregates for disease diagnosis. These assays are based on the self-replication properties of amyloid aggregates, which enable the amplification and detection of minute amounts of amyloid aggregates. My thesis focuses on the development of innovative amyloid amplification assays that enable not only the sensitive detection but also the absolute quantification of amyloid aggregates. I have developed amyloid amplification assays for three different amyloidogenic proteins including insulin, prion protein, and ↵-synuclein. In the first part of this thesis, I describe the development of a digital amyloid am-plification assay for the sensitive detection and absolute quantification of insulin amyloid aggregates. I used droplet-based microfluidics to encapsulate thousands of individual amplification reactions into small, uniform reaction compartments. Parallel, template-catalysed amplification led to the formation of large aggregates in droplets that initially encapsulated amyloid propagons. The number of propagons in the analyte was calculated from the fraction of aggregate-positive droplets of different dilutions of the analyte. The high number of replicate reactions yielded large, highly accurate data sets for rapid digital analysis using low reagent volumes. The assay proved to be a reliable and precise tool for the detection of single amyloid propagons, and for the absolute quantification of propagon numbers. The second part of my thesis focuses on the establishment and application of the real-time quaking-induced conversion (RT-QuIC) assay, an amyloid amplification assay for prion protein. RT-QuIC allows the detection of small amounts of the scrapie prion protein through seeded conversion of the cellular prion protein. In the first step, I implemented and validated the diagnostic RT-QuIC on cerebrospinal fluid samples from patients with suspected Creutzfeldt-Jakob disease. Samples from suspected cases were analysed by RT-QuIC, and the results were confirmed with autopsy results. Furthermore, I adapted the RT-QuIC assay to assess inactivation of prions adsorbed to surgical steel surfaces. Inactivation of prions adsorbed to surgical steel is crucial to reduce the potential risk of iatrogenic transmission of prion infectivity. A new formulation composed of hypochlorite, phosphate, silicate and phosphonate was identified as a potential decontaminant for prions bound to surgical steel. Finally, the assay was used to investigate prion self-replication in cerebellar organotypic cultured slices treated with neurotoxic anti-prion antibodies. The results showed that neurotoxic anti-prion antibodies do not induce self-replication of prions, but rather act downstream of prion self-replication. In the last part of my thesis, I describe the development of an ↵-synuclein amyloid amplification assay that can detect minimal amounts of ↵-synuclein aggregates. The assay has been used to investigate the inactivation of ↵-synuclein aggregates bound to stainless steel. Treatment with formic acid and sodium hypochlorite achieved effi-cient inactivation of ↵-synuclein propagons. Furthermore, the ↵-synuclein amyloid amplification assay was adapted to detect pathological aggregates in blood samples of affected patients. Different pretreatments were tested to enrich low-abundance aggregates in blood. Immunoprecipitation with specific anti-↵-synuclein antibodies was the most efficient treatment. Applying this treatment, I could demonstrate that samples from Parkinson’s disease patients showed low-level propagation activity in the amplification assay. The highly sensitive amyloid amplification assays developed in my thesis enabled the detection of minute amounts of pathological protein aggregates. A digital droplet-based amplification assay further allowed the determination of the absolute number of amyloid propagons in an analyte and the detection of single propagation units. These assays hold great potential for the early diagnosis of protein misfolding and aggregation diseases. In addition, the assays will help us to gain a more profound understanding of the molecular mechanisms underlying neurodegeneration.