Nanobody-basierter Sensor für die Zell-zu-Zell-Übertragung von α-Synuclein bei Morbus Parkinson

Nanobody-based sensor for cell-to-cell transmission of α-synuclein in Parkinson's disease

by Christoph Gerdes
Doctoral thesis
Date of Examination:2021-05-25
Date of issue:2021-05-18
Advisor:Prof. Dr. Silvio O. Rizzoli
Referee:Prof. Dr. Silvio O. Rizzoli
Referee:Prof. Dr. Tiago Fleming Outeiro
Referee:Prof. Dr. Martin Oppermann
Persistent Address: http://dx.doi.org/10.53846/goediss-8594

 

 

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Abstract

English

Aggregation and cell-to-cell transmission of α-Synuclein (αSyn) are hallmarks of several neurodegenerative diseases, including Parkinson's disease. In this work, it is shown that the nanobody NbSyn87, previously described to bind αSyn, also shows cross-reactivity for the proteasomal subunit Rpn10. This feature was exploited to design a new sensor for αSyn by fusing NbSyn87 to GFP and a nuclear localization signal. Therefore, when expressed in cells, the sensor is degraded by the proteasome in absence of αSyn but stabilized in presence of αSyn. This results in fluorescence signal fluctuations depending on the presence of intracellular αSyn. In this work, the biosensor is characterized in cells and shows that it is a sensitive sensor which specifically binds to human αSyn. Using the sensor, it is demonstrated that αSyn enters cells more efficiently when bound to a lipid layer. Finally, the sensor reveals the presence of transmittable αSyn in human cerebrospinal fluid. Thus, the sensor might be used to develop a diagnostic tool for synucleinopathies.
Keywords: Parkinson; alpha-Synuclein; transmission; nanobody; synucleinopathies; CSF; prion; synuclein; neurophysiology; neurodegeneration
 
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