aSyn is a phospholipid-binding protein with a role in vesicle trafficking and neurotransmitter release42. Tau is a microtubule-associated protein involved in microtubule stabilization and axonal outgrowth43,44. Both are soluble and natively unfolded proteins45,46, that present common mechanisms of transmission47, and are characterized by their pathological aggregation in neurodegenerative disorders. aSyn and Tau accumulation leads to the formation of oligomeric and fibrillar forms. A growing body of evidence points to an intertwined interaction between aSyn and Tau in pathology. Whilst patient-derived brain tissue exhibits co-occurrence of aSyn and Tau pathology in different neurodegenerative diseases, studies carried out in animal models show that aSyn and Tau interaction increases neurotoxicity. However, how aSyn and Tau influence each other, and how their interaction enhances neurodegeneration is still poorly understood.
One of the limitations of studying the consequences of aSyn-Tau interaction is the limited number of techniques available to monitor protein interaction in vivo. We applied the BiFC assay as a potential tool to visualize aSyn-Tau interaction. BiFC has been used by several groups aiming to obtain a direct visualization of protein oligomerization due to the relevance of oligomer formation in neurodegenerative disorders. In synucleinopathies and tauopathies, the presence and propagation of oligomeric forms of aSyn and Tau, respectively, correlates better than fibril formation with disease pathogenesis42,48.
Previously, the direct interaction between aSyn and Tau has been demonstrated in cell free assays25,27. Thus, in the present study we aimed to assess the ability of aSyn and Tau to interact with each other in a biologically relevant context, and to evaluate whether BiFC is a suitable technique to monitor aSyn-Tau interaction in vitro and in vivo.
BiFC was shown to be able to track aSyn-Tau (WT and P301L) interaction in vitro in HEK293 cells and in a neuronal-like cell line, SH-SY5Y cells. After confirming the functionality of this assay to detect aSyn-Tau interaction in vitro, in vivo injections in SNpc of WT mice and rats were performed. In mice, injections with AAV6 carrying the different BiFC constructs showed that aSyn-Tau interaction could be observed in vivo by BiFC. Besides the interaction between aSyn and Tau, we observed unexpected fluorescence when empty VN- was co-expressed with aSyn-VC or Tau-VC, which did not happen when the proteins linked to the N-terminal Venus half (VN-aSyn or VN-Tau) were co-expressed with the empty -VC construct. This phenomenon has been previously reported in the literature30,34,49.
Previous studies have explored the potential of BiFC to visualize protein interactions, however, some limitations of this assay need to be kept in mind. An imbalance in the levels of expression of the BiFC constructs has been long reported in the literature28–35, 37,40,49,50, suggesting an increase in the stability or decrease in the turnover of the VN- halves in comparison with -VC halves32. In our study we observed a similar phenomenon, that could potentially explain the propensity of the empty VN- half to interact with aSyn-VC and Tau-VC. Theoretically, the empty BiFC constructs (VN- and -VC) should not be able to complement when the interacting proteins are not present, yet, the presence of background fluorescence due to the interaction of the empty VN- construct has been previously described30,34,49.
The validity of the BiFC assay to monitor protein interaction has been tested by several groups. Robida et al. (2009) showed the ability of the BiFC assay to visualize protein interaction when this interaction had to be induced by the presence of a ligand49. In addition, point mutations in the amino acids involved in the interaction between proteins led to an inhibition of Venus complementation36,50−52. A recent study showed that even when the difference in the levels of expression of the BiFC constructs could lead to an increase in the self-aggregation of the overexpressed ones, the proteins of interest were able to physically interact with each other and this could be detected by BiFC32. Thus, the BiFC assay is suitable to determine protein-protein interactions but it is not sensitive enough to distinguish the degree of oligomerization between the interacting proteins32. This supports that the BiFC assay can be used to visualize aSyn-Tau interaction.
When VN-aSyn and Tau WT-VC AAVs were injected together in the SNpc of rats, we could observe that the co-expression of the proteins led to high levels of Tau phosphorylation at its S202 and T205 epitopes. Previous studies have shown the ability of aSyn to induce Tau phosphorylation1,20,21,25. Similarly, aSyn was phosphorylated at its pS129 epitope when co-expressed with Tau, although to a lesser extent.
In AD and PD, protein aggregation and neurodegeneration appear to follow a spatiotemporal progression53,54, that suggests cell-to-cell transfer of pathology55–57. This is supported by the spreading of aSyn aggregates from the host to healthy transplanted neurons observed in postmortem tissue of PD patients58,59. In our study, after co-injection of VN-aSyn and Tau-VC in rat SNpc, we could observe spreading of human aSyn from SNpc to the striatum, in contrast, there was much less transport of human Tau, which may be caused by the different size of aSyn and Tau molecules or by different levels of expression of the proteins.
We conclude that aSyn and Tau are able to interact in a biological setting, and that the BiFC assay is an effective tool to study aSyn-Tau interaction in vitro in cells and in different rodent models in vivo, providing a new resource for examining the pathological and physiological consequences of aSyn-Tau interaction. More work needs to be done to understand the impact of this interaction, and the consequences of its inhibition and/or facilitation in neurodegeneration.