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By Laura Torres-Garcia, Joana M. P. Domingues, Edoardo Brandi, Caroline Haikal, Janitha M. Mudannayake, Inês C. Brás, Ellen Gerhardt, Wen Li, Alexander Svanbergsson, Tiago F. Outeiro, Gunnar K. Gouras, and Jia-Yi Li

Excerpt from the article published in Scientific Reports volume 12, Article number: 2987 Published: 22 February 2022 DOI: https://doi.org/10.1038/s41598-022-06846-9

Editor’s Highlights

• α-synuclein (aSyn) is a phospholipid-binding protein with a role in vesicle trafficking and neurotransmitter release.
• Tau is a microtubule-associated protein involved in microtubule stabilization and axonal outgrowth.
• Both are soluble and natively unfolded proteins, that present a common mechanism of transmission, and are characterized by their pathological aggregation in neurodegenerative disorders
• The MAPT gene, which encodes for Tau protein; together with the SNCA gene, which encodes for aSyn protein, are major risk factors for the development of Parkinson’s disease (PD).
• aSyn and Tau are able to interact in a biological setting in pathology.
• aSyn and Tau interaction increases neurotoxicity. However, how aSyn and Tau influence each other, and how their interaction enhances neurodegeneration is still poorly understood.

Abstract

Parkinson’s disease (PD) and Alzheimer’s disease (AD) are characterized by pathological accumulation and aggregation of different amyloidogenic proteins, α-synuclein (aSyn) in PD, and amyloid-β (Aβ) and Tau in AD. Strikingly, few PD and AD patients’ brains exhibit pure pathology with most cases presenting mixed types of protein deposits in the brain. Bimolecular fluorescence complementation (BiFC) is a technique based on the complementation of two halves of a fluorescent protein, which allows direct visualization of protein–protein interactions. In the present study, we assessed the ability of aSyn and Tau to interact with each other. For in vitro evaluation, HEK293 and human neuroblastoma cells were used, while in vivo studies were performed by AAV6 injection in the substantia nigra pars compacta (SNpc) of mice and rats. We observed that the co-expression of aSyn and Tau led to the emergence of fluorescence, reflecting the interaction of the proteins in cell lines, as well as in mouse and rat SNpc. Thus, our data indicates that aSyn and Tau are able to interact with each other in a biologically relevant context, and that the BiFC assay is an effective tool for studying aSyn-Tau interactions in vitro and in different rodent models in vivo.

Introduction

Parkinson’s disease (PD) is the most common movement disorder and is characterized by the pathological accumulation of α-synuclein (aSyn) in the form of Lewy bodies (LB) and Lewy neurites (LN), and by the death of dopaminergic neurons in the substantia nigra in the midbrain¹. Alzheimer’s disease (AD) is the major form of dementia, and the accumulation of amyloid-β (Aβ) and Tau in Aβ plaques and neurofibrillary tangles (NFT), respectively, are pathological hallmarks of AD; however, Tau pathology correlates better with the cognitive decline associated with AD².

Genome-wide association studies (GWAS) have identified the MAPT gene, which encodes for Tau protein; together with the SNCA gene, which encodes for aSyn protein, as major risk factors for the development of PD^3,4. Furthermore, in human brains co-occurrence of aSyn and Tau pathologies has been described. About 30% of PD postmortem brains show NFTs^5,6, while LBs and LNs are detected in 60% of cases with AD^7,8. Moreover, aSyn aggregates are also found in Frontotemporal dementia (FTD)^9,10, Progressive supranuclear palsy (PSP)¹¹ and Down’s syndrome with AD^12,13. Likewise, Tau accumulation has been observed in Dementia with Lewy bodies (DLB), also called Diffuse Lewy body disease (DLBD)^14,15,16; and Parkinson’s disease dementia (PDD)^17,18. In particular, the co-occurrence of aSyn and Tau in amygdala has been reported in AD, PD, Down’s syndrome and DLB ^14,15,19,20.

Animal studies have shown that although Tau does not seem to be involved in aSyn spreading in the brain²¹, it plays an important role in synuclein pathology. In mice expressing human PD mutant A53T aSyn, Tau is required for the deficits in learning, memory and synaptic plasticity. A53T aSyn appears to induce Tau phosphorylation by glycogen synthase kinase-3 β (GSK3β) activation, which drives the missorting of Tau to dendritic compartments and an increase in the internalization of AMPA receptors (AMPARs), in the absence of clear neuropathology^22,23. Additionally, rats virally expressing human Tau (WT or P301L mutant) in substantia nigra pars compacta (SNpc) show vulnerability of dopaminergic neurons to Tau-induced neurodegeneration, and human Tau expression leads to a stronger amphetamine-induced rotational behavior than the expression of human aSyn (A30P or A53T mutant) alone in the same area²⁴. Similar observations have been described in a mouse model carrying the human K369I Tau mutation, in which impairment in the anterograde axonal transport induced by Tau results in loss of dopaminergic neurons and a Parkinsonian-like phenotype²⁵.

Moreover, aSyn appears to be essential for the phosphorylation of Tau in primary neurons after treatment with MPTP, a potent inducer of parkinsonism¹. Tau co-localizes and interacts with aSyn in aSyn aggregates²⁶ and both are found together by immunofluorescence microscopy in the axons of cultured primary hippocampal neurons²⁷, and in excitatory pre-synaptic terminals²⁸. Furthermore, cell free work has shown that aSyn and Tau induce each other’s fibrillization²⁹, and that aSyn is able to interact with Tau by binding of its C-terminal region to the microtubule binding domain (MTBD) of Tau^27,30. Thus, there is a growing body of evidence supporting that aSyn and Tau are able to interact and influence each other. However, how the interaction between aSyn and Tau enhances neurodegeneration is still poorly understood.

Bimolecular Fluorescence Complementation (BiFC) is a technique widely used to examine protein–protein interactions. It is based on the structural complementation of two halves of a fluorescent protein, allowing direct visualization of interacting proteins. In neurodegenerative diseases, this technique has been used to monitor the oligomerization of aSyn^{31,32,33,34,35}, Tau^34,36, Huntingtin^37,38, DJ-1^39,40, and TDP-43⁴¹ in vitro, as well as to study self-interaction of aSyn^42,43 and Tau⁴⁴ in vivo.

The aim of the present study was to assess the ability of aSyn and Tau to interact with each other, and to evaluate whether BiFC is a suitable technique to monitor aSyn-Tau interaction in vitro and in vivo. We concluded that aSyn and Tau are able to interact with each other and that the BiFC assay is an effective tool to study aSyn-Tau interaction in vitro and in vivo, providing a valuable tool for examining the pathological and physiological consequences of aSyn-Tau interaction, and facilitating the screening of potential drugs that could boost or inhibit this interaction.

Results

aSyn-Tau interaction in cell lines

The BiFC assay was chosen as a tool to evaluate aSyn-Tau interaction. aSyn (WT) and Tau (WT and P301L) were expressed fused to one of the halves of the Venus protein. After interaction of the two proteins of interest, the presence of both Venus halves in close proximity induces a conformational change in their structure that leads to their complementation and the emission of fluorescence that can be detected under a fluorescence microscope (Fig. 1A).

To examine whether aSyn-Tau interaction can be detected by the BiFC assay, we (co-) transfected HEK293 cells with different combinations of the BiFC constructs (Table S1). Single transfection of full-length Venus allowed us to determine the maximum level of Venus signal, whereas single transfection of aSyn-Venus confirmed that full-length Venus is able to emit fluorescence when linked to aSyn (Fig. 1B). On the other hand, co-transfection of the Venus halves (VN- and -VC) without aSyn or Tau linked proved that the Venus halves were not able to spontaneously complement with each other in the absence of aSyn and Tau (Fig. 1B). Similar results were obtained when VN-aSyn or VN-Tau WT were co-expressed with the empty -VC half. However, the expression of the empty VN- half together with aSyn-VC or Tau WT-VC gave rise to weak fluorescent signal (Fig. S1A).

Next, we used the BiFC assay to assess the self-interactions of aSyn, Tau WT and Tau P301L. HEK293 cells were co-transfected with paired BiFC constructs: VN-aSyn + aSyn-VC, VN-Tau WT + Tau WT-VC and VN-Tau P301L + Tau P301L-VC. These co-transfections resulted in the emergence of fluorescence-positive cells (Fig. 1B), reflecting the interaction of the proteins. Thus, we confirmed that self-interactions of aSyn, Tau WT and Tau P301L can each be observed by BiFC in HEK293 cells.

To evaluate aSyn-Tau (WT and P301L) interactions, HEK293 cells were co-transfected with aSyn and Tau (WT or P301L) linked to complementary Venus halves: VN-aSyn + Tau WT-VC, VN-Tau WT + aSyn-VC, VN-aSyn + Tau P301L-VC and VN-Tau P301L + aSyn-VC. In all the combinations, we observed the complementation of the Venus protein, reflecting the interaction of aSyn and Tau (Fig. 1C). The fluorescence obtained by complementation of the Venus protein was similar to the levels observed when evaluating the self-interactions of aSyn, Tau WT and Tau P301L. This demonstrates that aSyn-Tau (WT and P301L) interaction can be monitored in vitro by BiFC.

Prior work has shown differences in the levels of expression of the different BiFC constructs. To determine the protein levels upon transfection with the BiFC constructs in our study, HEK293 cells were co-transfected with different combinations of the BiFC constructs and 24 h post-transfection aSyn and Tau levels were evaluated. We observed that the protein levels of aSyn and Tau were higher when they were linked to the VN- half than when linked to the -VC half (Fig. 1D, S1B and C).

To test whether aSyn-Tau interaction can also be observed in a neuron-like cell, we (co-) transfected SH-SY5Y (human neuroblastoma) cells with the different combinations of the BiFC constructs (Table S1). As observed in HEK293 cells, the single transfection of Venus led to full Venus fluorescence, while aSyn-Venus expression demonstrated the ability of Venus to emit fluorescence when linked to aSyn (Fig. 2A). Additionally, the co-transfection of VN-, VN-aSyn or VN-Tau WT together with the empty -VC half showed no fluorescence, whilst we observed considerable fluorescence when co-expressing the empty VN- half together with aSyn-VC or Tau WT-VC (Fig. S2A). In SH-SY5Y cells, aSyn, Tau WT and Tau P301L were also able to self-interact (Fig. 2A), and we were able to detect complementation of Venus when aSyn and Tau (WT or P301L) were co-transfected (Fig. 2B). This shows that aSyn-Tau (WT and P301L) interaction can be observed in vitro by BiFC in a human neuroblastoma cell line.

To explore the dynamic processes of the interaction, Venus complementation in SH-SY5Y cells was examined by time-lapse, live-cell imaging every 30 min after transfection for 24 h. For each condition, a field of view (FOV) was chosen and tracked, and changes in fluorescence intensity over time in the cells within the FOV were measured. We observed initial fluorescence signal at different time points between conditions. Full-length Venus was the first in emitting fluorescence (~ 7.5 h), while fluorescence of aSyn-Venus took a longer time to appear (~ 11 h); however, both conditions showed similar intensity over time. Self-Interactions of aSyn, Tau WT, and Tau P301L showed similar initiation times (9–11 h) and progression, with the aSyn coupling exhibiting the strongest complemented fluorescence (Fig. S2B, upper graph). When aSyn and Tau (WT or P301L) were co-transfected, we could observe that the aSyn-Tau P301L couplings showed earlier initiation time but lower complemented fluorescence intensity than the aSyn-Tau WT couplings (Fig. S2B, lower graph).

Considering that the degree of interaction between the proteins in one cell could be influenced by the amount of plasmid(s) transfected into the cell and the levels of transgene expression, which could affect the dynamics of the interaction over time, we took a second approach to have a better picture of potential differences in the interaction between constructs. SH-SY5Y cells were (co-)transfected and after 24 h the percentage of fluorescent cells in relation to the total number of cells present in the FOV was quantified. This quantification showed no significant differences between aSyn, Tau WT, and Tau P301L when they were expressed by themselves or in combination. The percentage of fluorescent cells obtained after co-transfection of the different couplings was not significantly different to that obtained after single transfection of Venus or aSyn-Venus (Fig. 2C), while the fluorescence emitted by VN- + -VC coupling was undetectable.

Direct interaction of aSyn-Tau in mouse SNpc

Next, we asked whether the physiological environment of the living mouse brain would enable the detection of the aSyn-Tau interaction using BiFC. For that, AAV6 carrying different BiFC constructs (Table S2) were injected into the SNpc of 10–12-week-old WT mice, and 15 weeks post-injection complemented Venus fluorescence was evaluated. Low AAV titer was used to detect potential aSyn-Tau interactions without reaching toxicity. The 15 weeks time point was chosen due to that after AAV injection, the full expression of the transduced proteins in the brain takes up to 3–4 weeks. Moreover, previous publications showed self-interaction of Tau⁴⁴ or aSyn⁴² by BiFC complementation after 12 weeks, thus, supporting that 15–16 weeks would be a good time frame for studying aSyn-Tau interaction.

The expression of aSyn fused to full-length Venus provided the highest level of fluorescence. This expression was confined to a few neurons. As expected, and in agreement with the in vitro results, the expression of the Venus halves (VN- + -VC) not linked to interacting proteins did not produce any fluorescence in the injected area (Fig. 3). Similar results were observed when VN-aSyn or VN-Tau WT were co-expressed with the empty -VC half. However, the expression of the empty VN- half together with aSyn-VC or Tau WT-VC gave rise to a background residual fluorescence (Fig. S3A). Remarkably, the co-expression of aSyn and Tau (VN-aSyn + Tau WT-VC and VN-Tau WT + aSyn-VC) led to Venus complementation (Fig. 3), reflecting the interaction between aSyn and Tau in the SNpc of mice. Therefore, BiFC is shown to be a suitable technique to monitor aSyn-Tau interaction in vivo.

Further, we evaluated whether aSyn-Tau interaction could lead to toxicity in our system. For that, tyrosine hydroxylase (TH) immunolabelling was performed in mice injected with aSyn-Venus, VN- + -VC, VN-aSyn + Tau WT-VC, and aSyn-VC + VN-Tau WT (Fig. S3B). In general, the expression of the BiFC constructs did not seem to affect the SNpc; however, tissue damage associated with glass capillary tract injury was eventually observed, as can be noticed in aSyn-Venus injection (Fig. S3B). Moreover, rotarod and amphetamine-induced rotation tests were performed in the animals 15 weeks after the injection of the different AAV6. No significant differences were found between groups (data not shown), potentially due to the low number of neurons that expressed the BiFC constructs.

Direct interaction of aSyn-Tau in the rat SNpc

To corroborate that the BiFC assay can be used to visualize aSyn-Tau interaction in a different animal model, we injected AAV6 carrying VN-aSyn + Tau WT-VC, the aSyn-Tau combination that yielded the strongest interaction in our mouse system, in the rat SNpc. In this case, due to the lack of toxicity induced by AAV6 and based on the results obtained in mice, we increased the viral load 10 times. After 8 weeks, Venus complementation resulting from aSyn-Tau interaction was observed (Fig. 4A), confirming that the BiFC assay can be used in vivo in different rodent models. This complementation was observed both in SNpc (Fig. 4A) and in the striatum (Fig. S4), supporting that the interaction between aSyn and Tau is not only occurring in the soma of the transduced neurons but also at the terminal fields of their axons.

To characterize the state of aSyn and Tau, immunohistochemistry against phosphorylated forms of aSyn and Tau was performed. AT8 immunostaining showed that Tau was substantially phosphorylated at its S202 and T205 epitopes when co-expressed together with aSyn (Fig. 4A), consistent with prior work that showed that aSyn is able to induce Tau phosphorylation^1,22,23,27. In addition, aSyn was also phosphorylated at its S129 epitope. Interestingly, we observed differential co-localization between complemented (BiFC) Venus, AT8-positive and pS129-positive profiles. It appeared that most of the neuronal cell bodies positive for BiFC Venus are also positive for both AT8 and pS129 (Fig. 4A, white arrows), suggesting that both Tau and aSyn can be phosphorylated while they are interacting with each other. A few neuronal profiles with BiFC Venus exhibited either AT8- or pS129-positivity (Fig. 4A, orange arrows), implying that interacted Tau and aSyn may be phosphorylated to different extents. Furthermore, and unlike for Venus complementation, we did not observe phosphorylation of aSyn or Tau in the striatum.

Lastly, immunohistochemistry against total aSyn and total Tau was carried out to determine the distribution of the injected aSyn and Tau in the brain. Interestingly, marked propagation of the human aSyn was observed, indicative of transport of the protein from SNpc to the striatum. In contrast, the expression of human Tau remained mainly confined to the SNpc (Fig. 4B).

Discussion

aSyn is a phospholipid-binding protein with a role in vesicle trafficking and neurotransmitter release⁴⁵. Tau is a microtubule-associated protein involved in microtubule stabilization and axonal outgrowth^46,47. Both are soluble and natively unfolded proteins^48,49, that present common mechanisms of transmission⁵⁰, 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 pathogenesis^45,51.

Previously, the direct interaction between aSyn and Tau has been demonstrated in cell free assays^27,30. 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.

We showed that BiFC is able to track aSyn-Tau (WT and P301L) interaction in vitro in HEK293 cells and in a neuron-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 low titers of AAV6 vectors carrying the different BiFC constructs revealed that aSyn-Tau interaction could be observed in vivo by BiFC, without inducing toxicity. Interestingly, we observed a difference in the interaction between aSyn-Tau in relation to where the Venus halves were placed, resulting in greater interaction when VN-aSyn + Tau WT-VC were co-expressed than when aSyn-VC + Tau WT-VN were co-expressed. Next to the interaction between aSyn and Tau, we observed unexpected fluorescence when empty VN- was co-expressed with aSyn-VC or Tau WT-VC, which did not happen when the proteins linked to the N-terminal Venus half (VN-aSyn or VN-Tau WT) were co-expressed with the empty -VC construct. This phenomenon has been previously reported in the literature^33,37,52.

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 literature^{31,32,33,34,35,36,37,38,40,43,52,53}, suggesting an increase in the stability or decrease in the degradation of the VN- halves in comparison with -VC halves³⁵. Interestingly, Frey et al. (2020) addressed this imbalance in protein levels. They developed a bidirectional expression system that allowed for the inducible expression of both BiFC constructs (VN-aSyn and aSyn-VC) under the control of a single promoter. Nevertheless, even with this system, the imbalance in protein levels was seen, with VN-aSyn expression being higher than aSyn-VC expression. 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 is described^33,37,52.

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 ligand⁵². In addition, point mutations in the amino acids involved in the interaction between proteins led to an inhibition of Venus complementation^39,53,54,55. Moreover, 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 BiFC³⁵. 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 proteins³⁵. 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 phosphorylation^1,22,23,27. 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 progression^56,57, that suggests cell-to-cell transfer of pathology^58,59,60. This is supported by the spreading of aSyn aggregates from the host to healthy transplanted neurons observed in postmortem tissue of PD patients^61,62. In our study, after co-injection of VN-aSyn and Tau WT-VC in rat SNpc, we detected evidence consistent with transport of human aSyn from SNpc to the striatum, in contrast, there was much less transport of human Tau. However, the transport of Tau to the terminal fields of the axons was supported by the presence of Venus complementation in the striatum. Nevertheless, the difference in the propagation of aSyn and Tau in this study may be caused by, amongst others, the different size of aSyn and Tau molecules or by the 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. For example, future work in primary neuronal cultures could be of great value to better understand the mechanism(s) by which aSyn and Tau are able to interact with each other and the implications of this interaction in neuronal degeneration.