A nanobody-based fluorescent reporter reveals human α-synuclein in the cell cytosol

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