Monitoring Alpha-Synuclein – Tau Interactions In Vitro and In Vivo Using Bimolecular Fluorescence Complementation

Laura Torres-Garcia Lund University Joana M.P. Domingues Lund University Edoardo Brandi Lund University Caroline Haikal Lund University Inês C. Brás University Medical Center Göttingen Ellen Gerhardt University Medical Center Göttingen Wen Li Lund University Alexander Svanbergsson Lund University Tiago F. Outeiro University Medical Center Göttingen Gunnar K. Gouras Lund University Jia-Yi Li (  jia-yi.li@med.lu.se ) Lund University


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 midbrain 1 . Alzheimer's disease (AD) is the major form of dementia, and the accumulation of amyloid-β (Aβ) and Tau in Aβ plaques and neuro brillary tangles (NFT), respectively, are pathological hallmarks of AD; however, Tau pathology correlates better with the cognitive decline associated with AD 2 .
Genome-wide association studies (GWAS) have identi ed 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.
Animal studies have shown that although Tau does not seem to be involved in aSyn spreading in the brain 19 , it plays an important role in synuclein pathology. In mice expressing human PD mutant A53T aSyn, Tau is required for the de cits 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 20,21 . 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 amphetamineinduced rotational behavior than the expression of human aSyn (A30P or A53T mutant) alone in the same area 22 . 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 phenotype 23 .
Moreover, aSyn appears to be essential for the phosphorylation of Tau in primary neurons after treatment with MPTP, a potent inducer of parkinsonism 1 . Tau co-localizes and interacts with aSyn in aSyn aggregates 24 and both are found together by immuno uorescence microscopy in the axons of cultured primary hippocampal neurons 25 , and in excitatory pre-synaptic terminals 26 . Furthermore, cell free work has shown that aSyn is able to interact with Tau by binding of its C-terminal region to the microtubule binding domain (MTBD) of Tau 25,27 . Thus, there is a growing body of evidence supporting that aSyn and Tau are able to interact and in uence 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 uorescent protein, allowing direct visualization of interacting proteins. In neurodegenerative diseases, this technique has been used to monitor the oligomerization of aSyn 28-32 , Tau 31,33 , Huntingtin 34,35 , DJ-1 36,37 , and TDP-43 38 in vitro, as well as to study self-interaction of aSyn 39,40 and Tau 41 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.

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 uorescence that can be detected under a uorescence microscope ( Figure 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 fulllength Venus allowed us to determine the maximum level of Venus signal, whereas single transfection of aSyn-Venus con rmed that full-length Venus is able to emit uorescence when linked to aSyn ( Figure 1B). On the other hand, co-transfection of the Venus halves (VN-and -VC) without aSyn nor Tau linked proved that the Venus halves were not able to spontaneously complement with each other in the absence of aSyn and Tau ( Figure 1B). Similar results were obtained when VN-aSyn or VN-Tau were co-expressed with the empty-VC half. However, the expression of the empty VN-half together with aSyn-VC or Tau-VC gave rise to weak uorescent signal ( Figure 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 uorescencepositive cells ( Figure 1B), re ecting the interaction of the proteins. Thus, we con rmed that selfinteractions 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, re ecting the interaction of aSyn and Tau ( Figure 1C). The uorescence 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 ( Figure 1D and S1B).
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 uorescence, while aSyn-Venus expression demonstrated the ability of Venus to emit uorescence when linked to aSyn ( Figure  1A). Additionally, the co-transfection of VN-, VN-aSyn, VN-Tau or VN-Tau P301L together with the empty -VC half showed no uorescence, whilst we observed some uorescence when co-expressing the empty VN-half together with aSyn-VC, Tau-VC or Tau P301L-VC (Figure 2A and S2A). In SH-SY5Y cells, aSyn, Tau WT and Tau P301L were also able to self-interact (Figure 2A), and we were able to detect complementation of Venus when aSyn and Tau (WT or P301L) were co-transfected ( Figure 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 eld of view (FOV) was chosen and tracked, and changes in uorescence intensity over time in the cells within the FOV were measured. We observed initial uorescence signal at different time points between conditions. Full-length Venus was the rst in emitting uorescence (~7.5 h), while uorescence 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 uorescence ( Figure 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 uorescence intensity than the aSyn-Tau WT couplings ( Figure S2B, lower graph).
Considering that the degree of interaction between the proteins in one cell could be in uenced 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 uorescent cells in relation to the total number of cells present in the FOV was quanti ed. This quanti cation showed no signi cant differences between aSyn, Tau WT, and Tau P301L when they were expressed by themselves or in combination. The percentage of uorescent cells obtained after cotransfection of the different couplings was not signi cantly different to that obtained after single transfection of Venus or aSyn-Venus ( Figure 2C), while the uorescence 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 uorescence was evaluated.
The expression of aSyn fused to full-length Venus provided the highest level of uorescence. This expression was con ned 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 uorescence in the injected area ( Figure 3). Similar results were observed when VN-aSyn or VN-Tau were co-expressed with the empty -VC half. However, the expression of the empty VN-half together with aSyn-VC or Tau-VC gave rise to a background residual uorescence ( Figure S3). Remarkably, the co-expression of aSyn and Tau (VN-aSyn + Tau WT-VC and VN-Tau WT + aSyn-VC) led to Venus complementation ( Figure 3), re ecting 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.

Direct interaction 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 ( Figure 4A), con rming that the BiFC assay can be used in vivo in different rodent models.
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 ( Figure 4A), consistent with prior work that showed that aSyn is able to induce Tau phosphorylation 1,20,21,25 . 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 pro les. It appeared that most of the neuronal cell bodies positive for BiFC Venus are also positive for both AT8 and pS129 ( Figure 4A, white arrows), suggesting that both Tau and aSyn can be phosphorylated while they are interacting with each other. A few neuronal pro les with BiFC Venus exhibited either AT8-or pS129-positive ( Figure 4A, orange arrows), implying that interacted Tau and aSyn may be phosphorylated to different extents.
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 from SNpc to the striatum. In contrast, the expression of human Tau remained mainly con ned to the SNpc ( Figure 4B). Discussion aSyn is a phospholipid-binding protein with a role in vesicle tra cking and neurotransmitter release 42 .
Tau is a microtubule-associated protein involved in microtubule stabilization and axonal outgrowth 43,44 .
Both are soluble and natively unfolded proteins 45,46 , that present common mechanisms of transmission 47 , and are characterized by their pathological aggregation in neurodegenerative disorders. aSyn and Tau accumulation leads to the formation of oligomeric and brillar 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 in uence 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 bril formation with disease pathogenesis 42,48 .
Previously, the direct interaction between aSyn and Tau has been demonstrated in cell free assays 25,27 . 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 con rming 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 uorescence 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 literature 30,34,49 .
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 literature 28-35, 37,40,49,50 , suggesting an increase in the stability or decrease in the turnover of the VN-halves in comparison with -VC halves 32 . 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 uorescence due to the interaction of the empty VN-construct has been previously described 30,34,49 .
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 ligand 49 . In addition, point mutations in the amino acids involved in the interaction between proteins led to an inhibition of Venus complementation 36,50−52 . 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 BiFC 32 . 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 proteins 32 . 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 1,20,21,25 . 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 progression 53,54 , that suggests cell-to-cell transfer of pathology [55][56][57] . This is supported by the spreading of aSyn aggregates from the host to healthy transplanted neurons observed in postmortem tissue of PD patients 58, 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.

Materials And Methods
Animal work: mice and rats C57BL/6J mice and Sprague Dawley rats were purchased from Taconic (Ejby, Denmark). Animals were randomly assigned to each group. All experiments were approved and performed following the guidelines approved by the Malmö/Lund Ethics Committee, ethical permits number: M72-2016 and M73-2016. The reporting of the animal work performed in the manuscript follows the recommendations in the ARRIVE guidelines.

Generation of constructs and AVV production
The BiFC Tau constructs, VN-Tau (WT and P301L) and Tau-VC (WT and P301L), were generated by enzymatic restriction and ligation. Venus N-terminal (VN-) and Venus C-terminal (-VC) backbones were obtained from the VN-aSyn and aSyn-VC constructs previously described by Outeiro et al. (2008). Tau WT and Tau P301L sequences (0N4R isoform) were ampli ed from pRK5-EGFP-Tau (Addgene plasmid #46904) and pRK5-EGFP-Tau P301L (Addgene plasmid #46908) 60 , plasmids gift of Dr. Karen Ashe. In brief, Tau sequences were ampli ed by PCR (DreamTaq PCR Master Mix, Thermo Fisher Scienti c). The Venus halves (VN-and -VC) were used as a backbone. The backbones and the ampli ed Tau sequences were cleaved by AfIII (#ER0831, Thermo Fisher Scienti c) and XhoI (#ER0691, Thermo Fisher Scienti c) restriction enzymes, and ligated by incubation with T4 DNA ligase (#EL0014, Thermo Fisher Scienti c). The generated constructs were con rmed by sequencing. From these constructs, Adeno-Associated Virus Type 6 (AAV6) were produced in the AVV Vector Lab at the MultiPark core facility at Lund University.

Stereotaxic AAV Injections
Animals were unilaterally injected with a nal volume of 2 µl containing different combinations of the BiFC AAVs. Vector solutions were injected using a 5 µl Hamilton syringe tted with a glass capillary. The injection was performed manually in the case of mice, or by means of a pump with an infusion rate of 0.2 µl/min in rats. After delivery, the needle was left in place for 5 min before retraction.

Immunohistochemistry
Animals were deeply anaesthetized with iso urane, perfused rst with PB-buffer, followed by perfusion with 4% PFA, and the brains extracted. Brains were post-xed overnight and stored in 0.1 M PBS with 30% sucrose until sectioned. For mouse brain samples, 40 µm sections were mounted in polyvinyl alcohol mounting medium with DABCO (PVA-DABCO) containing DAPI (1:1000). Venus expression was acquired by using an inverted epi uorescence microscope Nikon Eclipse 80i under a 4x objective, and a Leica SP8 confocal microscope under a 40x objective.
For rat brain samples, 40 µm sections were washed in PBS-T, quenched in methanol and H 2 O 2 (9:1), washed in PBS-T, blocked in 5% BSA for 1 h, and incubated in aSyn (clone 211) or Tau-5 primary antibodies overnight. After incubation, sections were washed in PBS-T and incubated in anti-mouse HRP secondary antibody for 2 h, washed in PBS-T, incubated in ABC-solution (#PK-6100, Vector Laboratories), and washed in PBS-T and incubated in DAB solution (#SK-4100, Vector Laboratories). After that, sections were dehydrated and mounted in dibutyl phthalate in xylene (DPX; #06522, Sigma-Aldrich). For immuno uorescence, the sections were rinsed in TBS, pre-incubated in 10% Normal Donkey Serum (NDS) for 1 h and incubated overnight in pS129 and AT8 primary antibodies. Next, sections were rinsed with TBS, incubated in anti-rabbit and anti-mouse secondary antibodies for 1 h, rinsed with TBS and mounted in PVA-DABCO. After DAB staining, sections were scanned using an EPSON perfection V750 PRO with Silver Fast software. Fluorescence images were acquired in an Olympus BX53 microscope under a 10x objective. See list of antibodies in Table S3.

Statistical analyses
Data analysis was performed using GraphPad Prism v.9.2. Data were expressed as mean ± SD unless stated otherwise. Kruskal-Wallis test was performed to compare the ratio of BiFC positive cells per total amount of cells in FOV obtained 24 h after (co-) transfection of SH-SY5Y cells. Data was considered statistically signi cant when p-value < 0.05 (*p < 0.05, **p < 0.01).
The datasets generated during the current study are available from the corresponding authors on   aSyn-Tau interaction in mouse substantia nigra pars compacta (SNpc). BiFC interaction in mouse SNpc.
10-12-week-old mice were injected in SNpc with AAV6 carrying different BiFC constructs. After 15 weeks, Venus uorescence was evaluated. The expression of aSyn fused to full-length Venus provided the maximum level of uorescence expected. The interaction of aSyn and Tau was re ected by Venus complementation in VN-aSyn + Tau WT-VC and VN-Tau WT + aSyn-VC injected animals. As expected, the expression of the Venus halves not linked to proteins did not produce any uorescence in the injected area. Boxed area on Bright Field (B/F) images is magni ed to the right. Scale bar: 25 µm.

Figure 4
Molecular characterization of aSyn-Tau interaction in rat SNpc. aSyn-Tau interaction in rat SNpc. Eight weeks after co-injection of VN-aSyn and Tau WT-VC in rat SNpc immunohistochemistry was performed. A) Immunohistochemistry for phosphorylated epitopes of aSyn (pS129) and Tau (AT8) suggests that aSyn and Tau can be phosphorylated while they are interacting with each other. B) Distribution of aSyn and Tau after injection of VN-aSyn and Tau WT-VC in rat SNpc. aSyn distribution shows a marked spread of aSyn from the injected area to the striatum. In contrast, the expression of human Tau seems to be con ned to the SNpc. Scale bar: 50 µm

Supplementary Files
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