Periodic Reporting for period 1 - MicroSPARK (A Single-Molecule Technology for Resolving Chaperone Action in Neurodegenerative Diseases) Período documentado: 2019-04-01 hasta 2021-03-31 Resumen del contexto y de los objetivos generales del proyecto The Marie Skłodowska Curie Action “A Single-Molecule Technology for Resolving Chaperone Action in Neurodegenerative Diseases” (project acronym: MicroSPARK) was devoted to advance our technological capabilities of studying misfolding processes in neurodegenerative diseases and elucidate mechanisms of chaperone action and drug action. This was achieved through the first-time combination and integration of single molecule and microfluidics technologies, and their use in dissecting the action mechanism of chaperones.Neurodegenerative diseases, including Alzheimer’s and Parkinson’s disease, are increasingly prevalent disorders of our ageing society. These diseases arise from the formation of amyloidogenic protein aggregates. Molecular chaperones can counteract aggregate formation, but their molecular action mechanisms remain poorly understood. This Marie Skłodowska Curie Action addresses this challenge and implements the µSPARK technology to extend our understanding of the role of chaperones in the suppression of amyloid proliferation and in aggregate clearance, and introduces new means to sense amyloidogenic species for the development of therapeutics against neurodegenerative disease.The core advancement of this project is the implementation of microfluidic sorting devices with single molecule detection to address important questions related to protein misfolding diseases with a specific focus on chaperones and diagnostics. Specifically, microfluidic diffusional sizing combined with confocal fluorescence spectroscopy was used to study the disaggregation mechanism of the Hsc70 heat shock protein machinery. Moreover, free-flow electrophoresis was used in combination with laser-induced spectroscopy to resolve heterogeneities of oligomeric species created during aggregation reactions. In addition, the technological capabilities of the platform were used to establish a new diagnostic technology termed digital immuno-sensing assay (DigitISA). Trabajo realizado desde el comienzo del proyecto hasta el final del período abarcado por el informe y los principales resultados hasta la fecha The work of this Action was conducted in various stages. The first stage comprised the design, fabrication, and optimisation of microfluidic devices based on diffusional sizing and free-flow electrophoresis, and their integration with confocal spectroscopy. A laser-based single molecule fluorescence confocal setup was developed, built around an inverted microscope with a motorized stage, backport camera for imaging, and components required for single-photon counting and wavelength-sensitive fluorescence detection. In a second stage, the integrated microfluidics and single molecule platform was used to dissect the action mechanism of chaperones in curtailing α-synuclein aggregation. A manuscript on this work is being published (Schneider,* …, Krainer* et al., Nature Communications, accepted; biorxiv: https://doi.org/10.1101/2020.11.02.365825). Specifically, the disaggregation mechanism of the Hsc70 machinery was studied using the newly developed platform. We show that Hsc70 together with its co-chaperones DnaJB1 and Apg2 can completely reverse α-synuclein aggregation back to its soluble monomeric state. This reaction proceeds through first order kinetics where monomer units are removed directly from the fibril ends. These findings extend our mechanistic understanding of the role of chaperones in the suppression of amyloid proliferation and in aggregate clearance, and inform on possibilities and limitations of this strategy in the development of therapeutics against synucleinopathies. A further line of research was devoted to resolving oligomer heterogeneity underlying protein aggregation reactions. Free-flow electrophoresis in combination with confocal detection was used to resolve oligomeric species of the protein α-synuclein at the single-complex level in terms of population heterogeneity and physical properties (Krainer et al., in preparation). Finally, the technologies developed were additionally exploited in the development of DigitISA, a digital immuno-sensing approach with application in the diagnostics of oligomeric and fibrillar species in neurodegeneration. A patent on the technology has been filed and a manuscript is currently in revision (Krainer et el., in revision; biorxiv: https://doi.org/10.1101/2020.05.24.113498). The findings from this Action were communicated at international scientific conferences (e.g. Biophysical Society Meeting 2021) and published results were shared via modern Web 2.0 services (e.g. Twitter, Researchgate). Avances que van más allá del estado de la técnica e impacto potencial esperado (incluida la repercusión socioeconómica y las implicaciones sociales más amplias del proyecto hasta la fecha) This Action was designed to pioneer new technological approaches to better understand the mechanism of neurodegenerative diseases with the ultimate goal to find effective treatments and provide better diagnostic tools. The developments of this projects, with the implementation of microfluidic sorting devices with single molecule detection to address important questions related chaperone action, sets new grounds for the characterisation of hitherto experimentally inaccessible interactions of chaperones with amyloidogenic protein species. The implementation of the µSPARK technology has greatly increased our understanding of the biophysical mechanism by which chaperones curtail amyloid formation. Furthermore, the development of a new platform for biomolecular sensing of proteins, termed digital immuno-sensing assay (DigitISA) enables high sensitivity single-step detection and quantification of proteins in solution in a surface- and calibration-free manner—a thus far unmatched, yet coveted goal in biomolecular sensing and diagnostics. Parts of this work have been subject to a patent application. In conclusion, this Action has contributed to a better understanding of some of the world’s biggest challenges, such as healthy aging and precision medicine. Through the development of high-technology approaches, this Action has helped push the frontiers of biophysical research and has the potential to lead to new discoveries and innovations in biology and biomedicine. Figure 1. Overview of the main project results.