Aggregation and spreading of alpha-Synuclein (alpha Syn) are hallmarks of several neurodegenerative diseases, thus monitoring human alpha Syn (h alpha Syn) in animal models or cell cultures is vital for the field. However, the detection of native h alpha Syn in such systems is challenging. We show that the nanobody NbSyn87, previously-described to bind h alpha Syn, also shows cross-reactivity for the proteasomal subunit Rpn10. As such, when the NbSyn87 is expressed in the absence of h alpha Syn, it is continuously degraded by the proteasome, while it is stabilized when it binds to h alpha Syn. Here, we exploit this feature to design a new Fluorescent Reporter for h alpha Syn (FluoReSyn) by fusing NbSyn87 to fluorescent proteins, which results in fluorescence signal fluctuations depending on the presence and amounts of intracellular h alpha Syn. We characterize this biosensor in cells and tissues to finally reveal the presence of transmittable alpha Syn in human cerebrospinal fluid, demonstrating the potential of FluoReSyn for clinical research and diagnostics.
