The onset of Tau misfolding during early pathogenesis remains unidentified due to both the intrinsic disordered structure of the protein and the lack of suitable molecular tools. In order to overcome this limitation we are developing a set of engineered biosensors based on FRET. The wild type Tau biosensor, by virtue of its peculiar features, modulates its biophysical state in response to structural modifications occurring at early stages of neurodegeneration and it allows to monitor the conformational changes of Tau by FRET and the processing and mobility of cleaved fragments by FRAP. We monitored the conformation dynamics of tau-microtubules interplay and demonstrated that tau assumes a loop-conformation only when it is bound to microtubules. The disruption of tau-microtubules interaction and consequently the loss of tau loop-conformation detects one of the first events of tau misfolding occurring in neurodegeneration. Remarkably, by time lapse experiments we observed the rapid formation of tau aggregates similar to pathological aggregates in Alzheimer and tauopathies. Tau biosensors with mutations on residues involved in tau pathology have been subjected to this analysis in order to determine the contribution of each mutation. Recently several studies demonstrated that misfolded Tau species are able to propagate into the brain inducing the sprouting of the disease. In order to develop a sensitive tool to study the ability of infectious Tau species to propagate and to induce the misfolding into normal cells we set up a seeding activity assay by engineering a reporter cell line expressing the Tau biosensor. We demonstrated that the biosensor is a powerful readout in detecting the uptake of misfolded infectious tau species and their effect on endogenous Tau. In conclusion the tau biosensor is a suitable and sensitive tool to detect by imaging Tau conformational changes occurring during neurodegeneration, Tau aggregation and processing at early stages of pathology.
A new intramolecular biosensor reveals Tau conformational changes in pathological conditions
QUERCIOLI, VALENTINA;SIANO, Giacomo;ROVERE, MATTEO;VIEGI, Alessandro;CATTANEO, ANTONINO;DI PRIMIO, CRISTINA
2015
Abstract
The onset of Tau misfolding during early pathogenesis remains unidentified due to both the intrinsic disordered structure of the protein and the lack of suitable molecular tools. In order to overcome this limitation we are developing a set of engineered biosensors based on FRET. The wild type Tau biosensor, by virtue of its peculiar features, modulates its biophysical state in response to structural modifications occurring at early stages of neurodegeneration and it allows to monitor the conformational changes of Tau by FRET and the processing and mobility of cleaved fragments by FRAP. We monitored the conformation dynamics of tau-microtubules interplay and demonstrated that tau assumes a loop-conformation only when it is bound to microtubules. The disruption of tau-microtubules interaction and consequently the loss of tau loop-conformation detects one of the first events of tau misfolding occurring in neurodegeneration. Remarkably, by time lapse experiments we observed the rapid formation of tau aggregates similar to pathological aggregates in Alzheimer and tauopathies. Tau biosensors with mutations on residues involved in tau pathology have been subjected to this analysis in order to determine the contribution of each mutation. Recently several studies demonstrated that misfolded Tau species are able to propagate into the brain inducing the sprouting of the disease. In order to develop a sensitive tool to study the ability of infectious Tau species to propagate and to induce the misfolding into normal cells we set up a seeding activity assay by engineering a reporter cell line expressing the Tau biosensor. We demonstrated that the biosensor is a powerful readout in detecting the uptake of misfolded infectious tau species and their effect on endogenous Tau. In conclusion the tau biosensor is a suitable and sensitive tool to detect by imaging Tau conformational changes occurring during neurodegeneration, Tau aggregation and processing at early stages of pathology.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
