Targeting the N-terminus of α-synuclein Monomer Reduces Fibril-induced Aggregation in the Brain


 Background

Removing α-synuclein aggregates or preventing their formation constitutes a plausible strategy against Parkinson’s disease (PD). As we recently demonstrated, the β-wrapin protein AS69 binds an N-terminal region in monomeric α-synuclein (aSyn), interferes with fibril nucleation and reduces aSyn aggregates in vitro and in a fruit fly model of A53T aSyn toxicity. Here we tested whether AS69 could also reduce aSyn pathology in mammalian neurons and in mouse brain.
Methods

Primary mouse neurons were exposed to pre-formed fibrils (PFF) of human WT aSyn or PFF was injected into the striatum of A30P-aSyn transgenic mice to induce aSyn pathology. Densities of phospho-aSyn positive somatic inclusions and dystrophic neurites, degeneration of dopaminergic axon terminals in the striatum and the glial response were determined.
Results

PFF were readily taken up by primary mouse neurons, and AS69 did not alter PFF uptake. aSyn pathology, as determined by phospho-aSyn staining, was much more pronounced 72 h after PFF addition than after 24 h, and was reduced by AS69. In the striatum, PFF increased aSyn pathology, induced degeneration of dopaminergic axon terminals and glial activation at 90 days after PFF injection. The extent of terminal loss correlated with the density of dystrophic neurites, but not with the number of somatic inclusions. Co-injection of AS69 with PFF abrogated the induction of somatic inclusions and dystrophic neurites, reduced the loss of dopaminergic axon terminals and the reaction of astroglia, but not the effect on microglia.
Conclusion

AS69, an aSyn monomer-binding protein, reduces aSyn pathology and loss of dopaminergic terminals in primary neurons and in a mouse model. Therefore, our data suggests, that small aSyn-monomer binding proteins, such as AS69, could be promising new therapeutic approaches against Parkinson's disease.


Results
PFF were readily taken up by primary mouse neurons, and AS69 did not alter PFF uptake. aSyn pathology, as determined by phospho-aSyn staining, was much more pronounced 72 h after PFF addition than after 24 h, and was reduced by AS69. In the striatum, PFF increased aSyn pathology, induced degeneration of dopaminergic axon terminals and glial activation at 90 days after PFF injection. The extent of terminal loss correlated with the density of dystrophic neurites, but not with the number of somatic inclusions. Coinjection of AS69 with PFF abrogated the induction of somatic inclusions and dystrophic neurites, reduced the loss of dopaminergic axon terminals and the reaction of astroglia, but not the effect on microglia.
Conclusion AS69, an aSyn monomer-binding protein, reduces aSyn pathology and loss of dopaminergic terminals in primary neurons and in a mouse model. Therefore, our data suggests, that small aSyn-monomer binding proteins, such as AS69, could be promising new therapeutic approaches against Parkinson's disease.

Background
Parkinson disease (PD) and other synucleinopathies, such as dementia with Lewy bodies (DLB) and multisystem atrophy (MSA), are characterized by cytoplasmic aggregates of α-synuclein (aSyn). In PD and DLB, the major hallmarks of aSyn pathology are large inclusions in the neuronal soma termed Lewy bodies (LB) and dystrophic neurites with aSyn aggregates termed Lewy neurites (LN). Familial forms of PD can be caused by point mutations in the aSyn gene, including the A53T and A30P mutations, and by duplication of the aSyn locus. Polymorphisms in the gene encoding aSyn have been identi ed as risk factors for sporadic PD [1]. Preventing aSyn aggregation and removing aSyn aggregates are thus plausible neuroprotective strategies against synucleinopathies.
Molecular chaperones mediate the folding of newly produced or misfolded proteins and can prevent protein aggregation in both cellular and animal models of various neurodegenerative diseases [2]. While naturally occurring chaperones, e.g. HSP27 or HSP70, interact with aSyn via weak and transient interaction [3], AS69, an engineered β-wrapin, binds monomeric aSyn with high effectivity and high speci city [4]. AS69 wraps around a sequence region of monomeric aSyn that is critical for aggregation, comprising residues 37-54 [4][5][6], and stabilizes a β-hairpin conformation [4]. AS69 therefore represents a new paradigm in amyloid inhibition. We showed that the complex of AS69 with monomeric aSyn inhibits the proliferation of aSyn brils by interfering with primary and secondary nucleation processes [7].
Coexpression of AS69 in cultured cell lines reduced oligomerization and aggregation of both wild-type (WT) and A53T aSyn without altering the total levels of aSyn, suggesting that AS69 modulates the assembly of aSyn but does not induce its clearance. In a fruit y model of A53T aSyn toxicity, coexpression of AS69 reduced aggregation of aSyn in neurons and rescued locomotor de cit resulting from neuronal aSyn expression.
In order to validate these ndings in mammalian neurons, we now tested the effect of AS69 on aSyn aggregate formation induced by the addition of pre-formed brils (PFF) made from recombinant human WT aSyn [8]. Furthermore, we tested AS69 in the more complex biological environment of a mammalian brain using transgenic mice expressing in neurons human aSyn with the A30P mutation [9]; these mice develop pathology spontaneously, but late in life. To accelerate aSyn pathology, we therefore injected aSyn PFF into the striatum. AS69 reduced the extent of aSyn pathology, the loss of dopaminergic axon terminals and the reaction of glial cells.

Recombinant proteins and bril formation
Human WT α-synuclein protein and AS69 were produced in bacteria and puri ed as previously described [10]. In the nal puri cation step, WT aSyn was eluted from a Superdex 75 column (GE Healthcare) equilibrated in PBS buffer. PFF were generated using a standard protocol [8]. In brief, 500 µl of a 5 mg/ml solution of aSyn in PBS were incubated in 1.5 ml microcentrifuge tubes on a Thermomixer (Eppendorf) sealed with para lm for 7 days at 37 °C under constant agitation (1000 rpm). Fibril formation was con rmed by Thio avin T uorescence and atomic force microscopy.
Animals and surgery C57BL6/J-Thy1-A30P-α-synuclein mice [9] have been used in previous studies [11][12][13]. They were bred as homozygous, housed and handled in a pathogen-free animal facility at 20-24 °C with a 12 h light/dark cycle, food and water ad libitum, in accordance with guidelines of the Federation for European Laboratory Mice were identi ed by three-digit numbers and assigned to one of three experimental groups using random numbers by a technician not involved in the design of the study. The injected solution was prepared freshly on each experimental day from frozen aliquots of the following three stock solutions: sterile phosphate buffer solution (PBS), PFF in PBS at a concentration corresponding to 5 mg/ml aSyn protein, 404 µM AS69 in PBS. After thawing, PFF were diluted in PBS in order to obtain the same PFF concentration both with and without AS69. The nal solutions contained (1) PBS only, (2) 1.4 mg/ml αsynuclein equivalent PFF, (3) 1.4 mg/ml α-synuclein equivalent PFF + 98 µM AS69. (98 µM AS69 is roughly equimolar to 1.4 mg/ml α-synuclein.) After dilution, solutions were sonicated at room temperature with 10% power output and 60 pulses of one second each (300 VT; Biologics, Inc., Manassas, VA).
Stereotaxic injection into the right striatum (AP: 1; ML: 1.5 relative to Bregma; DV: 1.55 from dura) and tissue preparations were performed essentially as described earlier [12]. Brie y, 47-57 week old mice (mixed gender) received 2.5 µl of one of the three solutions (PBS, PFF, PFF + AS69) with a ow rate of 0.2 µl/min. Mice were sacri ced 90 days after the injection. Brains were extracted, xed at 4 °C in 4% paraformaldehyde in PBS for 24 hours and cryoprotected in 30% sucrose in PBS. Free-oating, 30 µm serial coronal sections were collected with a Cryostat (Leica Microsystems, Germany). Brain slices were stored at − 20 °C (30% glycerol, 30% ethylene glycol in 0.1 M PB) until use.

Primary neuronal culture
Primary neuronal cultures were prepared from P1-3 C57BL6/J mouse pups of mixed sex. Brie y, after dissection and trypsinisation, dissociated neurons were plated onto poly-L-ornithine (Sigma) coated glass coverslip (100 000 cells per well in 24-well dishes), and maintained in Neurobasal A medium containing B27 (2%, Invitrogen), glutamax (0.5 mM, Invitrogen) and antibiotics [14]. One third of the medium was changed on every third day, and from the second change, no antibiotics was added. On DIV 12, neurons were incubated with BSA (1 µg/ml, protein control), 50 nM AS69, PFF corresponding to 50 nM aSyn monomer or the same concentration of PFF + 50 nM AS69. Neurons were analysed on day 13 (24 h after adding PFF) or day 15 (72 h after adding PFF). Experiments were repeated 3-4 times (n = 3-4).

Analysis of aSyn pathology, striatal ber density and gliosis
To quantify the extent of aSyn pathology in the striatum, we used the StereoInvestigator software (MicroBright eld Bioscience, Williston, VT) and the optical fractionator method. Every 6th slice of the striatum was stained. In each slice, a grid of 200 µm × 200 µm was placed randomly over the striatum outlined using the 2.5x objective. At each intersection of the grid, a 100 µm × 100 µm counting frame was inspected manually for phospho-aSyn positive somatic inclusions and dystrophic neurites using a 63x oil objective and an Axio Imager 2 microscope (Carl Zeiss Vision, Göttingen, Germany). The investigator carrying out this analysis was not involved in the surgery and unaware of the injection obtained by each animal (identi ed by three-digit numbers). Counts re ect the number of items estimated by the stereology procedure for each animal.
For quanti cation of the density of striatal TH-positive bers, uorescence 8-bit images were acquired as z-stack ( ve slices, 1 µm step) using a 60x oil objective (NA 1.35) and an IX81S1F microscope (Olympus, Hamburg, Germany). A maximum intensity projection over z was performed and TH-positive bres delineated using the auto-threshold function of ImageJ (1.47v; NIH, Bethesda, MD, USA). Fiber density was expressed as percent area. Three section per animal, and ve images per section were analysed resulted in a hierarchically nested design. A generalized linear mixed model was applied [13].
For quanti cation of gliosis, uorescent images were acquired using a 20x objective (NA 0.8) with an Axio Imager 2 microscope (Zeiss). After adjusting the threshold on the single images (separate for GFAP and for Iba1; ImageJ), area fraction was determined from 2 sections per animal and from 10 images per section (see above).
The number of dopaminergic neurons in the substantia nigra was quanti ed as previously [16]: THpositive neurons in the SNc of the right hemisphere were stereologically counted using the optical fractionator method (StereoInvestigator) in every third section (counting frame: 100 × 100 µm, grid size; 200 × 200 µm; oil immersion 63x objective, NA 1.25).
To measure aSyn uptake and pathology, images of primary cultures were acquired with Zeiss Axio Imager 2 using a 100x oil immersion objective (NA 1.4), with a monochrome camera (Axiocam 705 mono, Zeiss).
For each experiment and experimental group, a minimum of 10 neurons were selected randomly. Exposure time was kept constant for each staining across all experimental groups. To outline neurons, a mask was created based on the MAP2 channel. In this mask, the area fraction of (a) phospho-aSyn and (b) human aSyn staining was determined, using ImageJ 1.52 h. Images are pseudo coloured for better visualization.

Statistical analysis and data visualisation
For cell cultures, "n" was set to the number of individual experiments, and for the animal experiments, "n" was set to the number of mice. For each experimental condition and experiment, or for each animal, values were summarized by determining the mean. Data are presented as markers for each condition within a single experiment, and for each animal and as mean ± standard deviation. Comparisons were performed one-way or two-way ANOVA using GraphPad Prism 5 (Version 5.01). p < 0.05 was considered signi cant. p values are depicted as * p < 0.05; ** p < 0.01.

AS69 decreases intracellular aSyn pathology in primary neurons
AS69 is a small (15 kDa) protein that interferes with aSyn aggregation in a substoichiometric way [4,7]. The mechanism includes the inhibition of secondary nucleation, which is a critical part of the prion-like behaviour of aSyn and de ned as the acceleration of aggregation by the propagation of template aggregates [PMID: 29978862]. To validate our previous ndings from cell lines and fruit ies, we used a primary neuronal model of secondary nucleation, where pre-formed brils (PFF) of human WT aSyn are added to the neuronal culture medium [8]. In this model, PFFs are readily taken up by neurons, and induce aggregation of endogenous (mouse) aSyn [17][18][19]. Since AS69 does not dissociate aSyn brils [4,7,10], we added AS69 as recombinant protein to the culture medium. BSA was used as negative control.
First, we detected intracellular human aSyn 24 hr after PFF treatment (Fig. 1A, B). In accordance with previous work cited above, this nding con rms that PFF are readily taken up by primary mouse neurons. In addition, we determined the extent of aSyn pathology by staining for phospho-aSyn (Fig. 1A, C).
Whereas staining for human aSyn increased most strongly within the rst 24 h, staining for phosphosynuclein increased mainly between 24 and 72 h. This is consistent with the hypothesis that seeding of aSyn pathology by PFF takes time. AS69 did not alter the amount of human aSyn at 24 h (Fig. 1B), indicating that AS69 does not affect aSyn uptake. This is in agreement with our previous nding that AS69 does not directly affect aSyn brils [4,7]. AS69 did reduce the extent of phospho-aSyn pathology at 72 h (Fig. 1C), consistent with our previous nding that AS69 reduces secondary nucleation. AS69 also reduced the staining for human aSyn at 72 h (see discussion).
AS69 was added extracellularly and changed intracellular aSyn pathology. To address whether AS69 can be taken up by neurons, we stained for AS69 in cells exposed to AS69 but not PFF. Indeed, AS69 was found inside the neurons (Fig. 1A, BSA + AS69 group), indicating that it can be taken up independently from PFF. Of note, AS69 alone did not induce aSyn pathology (Fig. 1C).
The intracellular area positive for human aSyn staining increased strongly between 24 and 72 h (Fig. 1B). This is not explained by the ndings discussed so far. We cannot rule out that a subtle effect of AS69 on PFF uptake ampli es over time. However, human aSyn PFF induces the aggregation of endogenous mouse aSyn, and the deposition of phospho-aSyn-positive inclusions is rather mouse aSyn-driven [20]. Therefore the difference in human aSyn staining at 72 h is better explained by the fact that the particles revealed by human aSyn staining are not only composed of human aSyn but contain endogenous mouse aSyn as well. In other words, the area of human aSyn staining does not faithfully report the amount of intracellular human aSyn protein but rather the area of aSyn pathology containing human aSyn. If the increase in human aSyn staining between 24 and 72 h results from expansion of pathology containing human aSyn, the smaller area with AS69 is explained by reduced seeding of aSyn pathology with AS69.
In addition, fewer and smaller aSyn aggregates are easier to degrade, so reduced seeding of aSyn pathology by AS69 could also facilitate clearance of aSyn aggregates.

AS69 reduces PFF-induced α-synuclein pathology in vivo
Taken together, AS69 reduced the amount of aSyn pathology in primary neurons but did not interfere with PFF uptake. To expand on this nding, we asked whether AS69 could also reduce PFF-induced aSyn pathology in mice. To induce a robust aSyn pathology, we injected the same human WT aSyn PFF into the striatum of 47-57 week old mice with neuronal expression of human A30P-αSyn [9]. aSyn transgenic mice were used because in these mice neurons express more aSyn, which facilitates initiation of aSyn pathology. Brains were analysed 90 days after the injection (Fig. 2A). To reveal striatal aSyn pathology induced by PFF injection, we visualized phospho-aSyn positive structures (Fig. 2B) and discriminated two phenotypes: (1) Somatic Accumulations of phospho-aSyn staining ("SA", solid arrows in Fig. 2B) and (2) Dystrophic, phospho-aSyn positive Neurites ("DN", open arrows in Fig. 2B). These changes are reminiscent of the somatic Lewy bodies and Lewy neurites in human brain, respectively. Numbers of SA and DN in each striatum were determined by stereology.
Even in vehicle-injected mice we saw a relevant number of striatal SA (Fig. 2B, C) and DN (Fig. 2B, D) and attribute this to the neuronal overexpression of A30P-aSyn in this mouse line. In PFF-injected striatum we observed more SAs (Fig. 2C) and DNs (Fig. 2D) than in the contralateral striatum, indicating that PFF induced additional aSyn pathology. Of note, the extent to which PFF injection increased phospho-aSyn pathology differed between SA and DN: The ratio between the injected and non-injected hemispheres was signi cantly different from the control for DN but not for SA (Fig. 2C, D). In accordance with our in vitro ndings (Fig. 1C), co-injection of AS69 with the PFF prevented the induction of aSyn pathology: With AS69, the number of SA and DN was not different between the ipsi-and contralateral hemispheres (Fig. 2C, D), and the ratio between both hemisphere did not differ from control ( Supplementary Fig. 1A, B).

PFF-induced degeneration of dopaminergic axon terminals
A30P-α-synuclein transgenic mice do not show spontaneous degeneration of dopaminergic neurons in the substantia nigra or degeneration of their axon terminals in the striatum [11]. Yet, the injection of PFF into the striatum reduced the density of dopaminergic axon terminals by about 20% (Fig. 3A, B; absolute densities of each hemispheres are shown in Supplementary Fig. 1C). Co-injection of AS69 prevented the PFF-induced degeneration of dopaminergic axon terminals (Fig. 3A, B). The number of dopaminergic cell bodies in the substantia nigra pars compacta was not signi cantly altered by PFF injection into the striatum or co-injection of AS69 (data not shown).
Although it is widely accepted that aSyn pathology is a major hallmark of PD and driver of neurodegeneration, it is still a matter of debate which type of aSyn pathology better correlates with neurotoxicity and functional impairment [21]. Therefore, we correlated on an animal basis the extent of SA and DN with the density of dopaminergic axon terminals. Interestingly, while the increase of round, somatic, Lewy-body-like SA showed no correlation with dopaminergic ber loss (Fig. 3C), we observed a strong correlation between the number of DNs and the reduction in dopaminergic axon terminals (Fig. 3D). This difference suggests that neuritic aSyn pathology is more closely linked to the degeneration of dopaminergic axon terminals than somatic accumulations.

Response of glial cells to PFF injection
To determine to what extent glial cells respond to PFF injection, we stained striatal sections for the microglial marker Iba1 or the astroglial marker GFAP (Fig. 4A). PFF injection induced a signi cant activation of both microglia (Fig. 4B) and astroglia (Fig. 4C). Injection of PBS alone did not produce a signi cant glial activation, con rming that that the observed effects can indeed be attributed to the PFF and are not a nonspeci c response to the intracerebral injection. AS69 did not moderate the Iba1 response (Fig. 4B). Accordingly, the Iba1 response did not correlate with the reduction in dopaminergic bers (supplemental Figure S1G), or with the extent of DN and SA pathology (supplemental Figure S1H, I). Activation of astroglia, in contrast, was signi cantly reduced by AS69 (Fig. 4C). Moreover, the extent of astroglial activation correlated inversely with the density of dopaminergic bers (Fig. 4D) and correlated positively with the relative density of DNs (Fig. 4E). The extent of astroglial activation did not correlate with the density of SA (supplemental Fig. 1F). Discussion aSyn aggregates are the main brillary components of Lewy neurites and Lewy bodies, and targeting those aggregates might hold therapeutic potential. In our previous studies [4,7,10] we reported on a novel strategy to inhibit aSyn nucleation by using a high a nity monomer binding molecule, AS69. Here, we investigated the effect of AS69 in PFF-based in vivo models of synucleinopathies. We show, that AS69 did not alter PFF uptake in primary neurons, but it reduced PFF-induced aSyn pathology. In mice, AS69 ameliorated PFF-induced aSyn pathology in the striatum, loss of dopaminergic axon terminals and astroglia activation.
Chronic overexpression of human A30P aSyn and acute injection of PFF from human WT aSyn trigger morphologically distinct types of aSyn pathology (Fig. 2C, D). We propose that neurons with chronic overexpression of human A30P aSyn can still maintain retrograde transport and collect aSyn aggregates in perinuclear accumulations (SAs). Indeed, in the A30P mice without PFF injection, basal aSyn pathology is mostly restricted to somatic accumulations (SAs, reminiscent of LBs) with only few dystrophic neurites (DNs, reminiscent of LNs), consistent with earlier studies [22]. These mice do not show a loss of dopaminergic somata in the substantia nigra, degeneration of dopaminergic axon terminals in the striatum or glial activation (Figs. 2B, 3A and reference [11]), but their neurons are vulnerable [13,[22][23][24].
Toxic insults, such as MPTP or PFF disrupt retrograde transport and damage neurite integrity. Accordingly, injection of PFF into A30P mice induced a robust increase in aSyn pathology, consistent with previous work for other aSyn transgenic mice [25,26]. The PFF-induced increase in the number of DN was much more pronounced than for SA (Fig. 2C,D). This formation of DN may result from excessive seeding of aSyn aggregates in neurites and from impaired transport of aSyn aggregates to the soma. Damaged neurites can explain the loss of dopaminergic axon terminals in the striatum, a causal relationship is supported by the tight correlation of DN with dopaminergic ber loss (Fig. 3D). In the striatum, dopaminergic axon terminals are particularly vulnerable because they are far from the neuronal soma, and because dopamine further increases aSyn aggregation [27,28]. Still, we expect that not all DN we observed are dopaminergic, and that PFF also trigger aSyn aggregation in non-dopaminergic neurites.
AS69 reduced DN (Fig. 2D) and we propose that this underlies the higher density of remaining axon terminals (Fig. 3B).
Neurodegeneration and the presence of aSyn pathology are often accompanied by activation of glial cells [29,30]. We found a moderately increased microglia activation in aSyn transgenic animals (Fig. 4A), consistent with previous studies using the same mouse line [30]. PFF injection led to an activation of both glia types, but the extent of microglia activation did not correlate with the amount of synuclein pathology (DAs or SAs) or with neuronal damage, represented by dopaminergic ber loss (Supplementary Figure G-I). This suggests that in this animal model, microglia do not have a direct role in neuronal damage, even though aggregated aSyn is a potent activator of microglia in other paradigms [31,32]. In contrast, astroglia was not activated in controls, but increased with PFF (Fig. 4A). The extent of astroglia activation correlated with the number of DN and with the density of dopaminergic bers, suggesting an involvement of astroglia in PFF-induced neuronal damage (Fig. 4D, E); further work will be required to determine the role of astroglia activation. Consistent with the involvement of astroglia but not microglia in aSyn dependent damage, AS69 reduced astrogliosis but did not affect microglia activation.
What is the cellular mechanism of AS69? In mouse primary neurons exposed to PFF with either BSA or AS69 for 24 h, we detected the same amount of intracellular human aSyn (Fig. 1B). We therefore assume that AS69 does not in uence uptake of PFF, consistent with our previous nding that AS69 does not induce disassembly of higher order aSyn species [4]. We also showed that neurons take up AS69 (Fig. 1A), similar to other proteins [27], allowing the protective effects of AS69 to be explained by intracellular events. With AS69, the amount of phospho-aSyn 72 h after adding PFF to cultured neurons was signi cantly smaller (Fig. 1C), consistent with the lower number of DN in mice (Fig. 2D) and in drosophila neurons (Agerschou et al., 2019). Based on our previous ndings that the complex of AS69 and aSyn inhibits secondary nucleation and aggregate ampli cation [7] the most probable mechanism of AS69 is to prevent seeding of aSyn pathology by binding intracellular aSyn monomers.

Conclusions
Taken together, we demonstrated that the quanti cation of DNs, dopaminergic axon terminals and astrogliosis are sensitive measures of neuronal damage in our animal model. AS69 reduced the induction of aSyn pathology by PFF, reduced the loss of dopaminergic bers and decreases astrogliosis. Prevention of seeding of aSyn pathology by monomer sequestration might therefore represent a plausible therapeutic strategy for synucleinopathies.