RESUMEN

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

Asunto(s)

Citosol/metabolismo , Proteínas Luminiscentes/metabolismo , Anticuerpos de Dominio Único/metabolismo , alfa-Sinucleína/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Células Cultivadas , Citosol/química , Femenino , Fluorescencia , Células HEK293 , Humanos , Proteínas Luminiscentes/química , Proteínas Luminiscentes/genética , Masculino , Microscopía de Fluorescencia por Excitación Multifotónica , Persona de Mediana Edad , Neuronas/citología , Neuronas/metabolismo , Ratas Wistar , Anticuerpos de Dominio Único/genética , alfa-Sinucleína/líquido cefalorraquídeo , alfa-Sinucleína/genética , Proteína Fluorescente Roja