Snca-GFP knock-in mice reflect patterns of endogenous expression and pathological seeding

Alpha-synuclein (aSyn) participates in synaptic vesicle trafficking and synaptic transmission, but its misfolding is also strongly implicated in Parkinson’s disease (PD) and other neurodegenerative disorders known as synucleinopathies where misfolded aSyn accumulates in different regions of the central and peripheral nervous systems. Although increased aSyn expression levels or altered aggregation propensities likely underlie familial PD with SNCA amplification or mutations, the majority of synucleinopathies arise sporadically, indicating that disease can develop under normal levels of wildtype aSyn. We report here the development and characterization of a mouse line expressing an aSyn-GFP fusion protein under the control of native Snca regulatory elements. Regional and subcellular localization of the aSyn-GFP fusion protein in brains and peripheral tissues of knock-in (KI) mice are indistinguishable from that of wildtype littermates. Importantly, similar to wildtype aSyn, aSyn-GFP disperses from synaptic vesicles upon membrane depolarization, indicating that the tag does not alter normal aSyn dynamics at synapses. In addition, intracerebral injection of aSyn pre-formed fibrils into KI mice induced the formation of aSyn-GFP inclusions with a distribution pattern similar to that observed in wildtype mice, albeit with attenuated kinetics due to the GFP tag. We anticipate that this new mouse model will facilitate in vitro and in vivo studies requiring in situ detection of endogenous aSyn, therefore providing new insights into aSyn function in health and disease. Significance Statement Alpha-synuclein (aSyn) participates in synaptic vesicle function and represents a major component of the Lewy pathology found in Parkinson’s and related neurodegenerative diseases. The function of aSyn and the sequence of events leading to its aggregation and neurotoxicity are not fully understood. Here we present a new mouse model in which Enhanced Green Fluorescence Protein (GFP) has been knocked-in at the C-terminal of the Snca gene. The resulting fusion protein shows identical expression and localization to that of wildtype animals, is functional, and is incorporated into pathological aggregates in vitro and in vivo. This new tool allows for monitoring aSyn under a variety of physiological and pathological conditions, and may uncover additional insights into its function and dysfunction.


Introduction
Alpha-synuclein (aSyn) is a protein prominently expressed in neurons and enriched at presynapses (Maroteaux et al., 1988). Although its precise functions are not fully understood, collective evidence suggests that aSyn regulates synaptic vesicle trafficking and fusion (Burre et al., 2018;Logan et al., 2017;Nemani et al., 2010;Wang et al., 2014). aSyn also constitutes the major component of Lewy bodies and Lewy neurites, intraneuronal inclusions characteristic of Parkinson's and a group of neurodegenerative diseases known as synucleinopathies (Bendor et al., 2013;Burre et al., 2018;Goedert et al., 2013). Although mutations or amplification of the aSyn gene were linked to familial PD (Koros et al., 2017;Kruger et al., 1998;Polymeropoulos et al., 1997;Schneider and Alcalay, 2017;Singleton et al., 2003;Zarranz et al., 2004) the majority of PD cases are sporadic and develop in the presence of normal levels of wildtype (wt) aSyn. 5 Several aSyn animal models have been generated to study aSyn function and pathobiology (Visanji et al., 2016). Accumulation of aggregated aSyn, loss of dopaminergic neurons, and behavioral impairments have been reported in multiple lines.
However, the majority of these models rely on the overexpression of human aSyn, often bearing disease-associated mutations, under the control of a heterologous (i.e. non-Snca) promoter, and in the presence of endogenous mouse aSyn expression (Emmer et al., 2011;Giasson et al., 2002;Kahle et al., 2001;Masliah et al., 2000;Matsuoka et al., 2001;Tofaris et al., 2006;van der Putten et al., 2000). These features might confound data interpretation, especially in relation to sporadic PD. These models also poorly recapitulate the prion-like propagation of pathological aSyn suggested by the staged distribution of aSyn deposits in diseased human brains (Braak et al., 2003;Goedert et al., 2013;Jucker and Walker, 2018). Recent studies demonstrating that aSyn pathology formation and spread can be initiated in wt mice following inoculation of recombinant aSyn preformed fibrils (PFFs) (Luk et al., 2012;Masuda-Suzukake et al., 2014;Rey et al., 2018) or human brain derived aSyn aggregates (Masuda-Suzukake et al., 2013;Peng et al., 2018;Recasens et al., 2014) further support this hypothesis, thus enabling the modeling of these processes without aSyn ectopic expression. Exposure to PFFs also induces aSyn pathology in primary neurons, allowing for cellular and molecular characterizations (Volpicelli-Daley et al., 2011). These models provide additional structural and temporal resolution for observing aSyn pathogenesis, yet they do not allow to monitor aSyn in realtime. To this aim, two mouse models overexpressing human aSyn tagged with GFP have been reported (Hansen et al., 2013;Rockenstein et al., 2005). Transient overexpression of aSyn-GFP has also been used in primary neurons (Fortin et al., 2005;McLean et al., 6 2001; Volpicelli-Daley et al., 2014). These models have provided valuable insights about the trafficking of aSyn at synapses (Fortin et al., 2005), aSyn aggregate formation and development (Osterberg et al., 2015;Unni et al., 2010) and their link to synaptic/axonal dysfunction (Scott et al., 2010;Volpicelli-Daley et al., 2014) and cell death (Osterberg et al., 2015).
However, aSyn is differentially expressed in specific neuron subtypes (Hawrylycz et al., 2012;Taguchi et al., 2016;Uhlen et al., 2015) and its function and propensity to aggregate is dependent on expression levels (Logan et al., 2017;Luna et al., 2018;Nemani et al., 2010;Scott and Roy, 2012), thus making the use of the endogenous promoter highly desirable. We therefore created a mouse in which GFP was knocked-in at the carboxy-terminal of the endogenous Snca gene. The resulting mouse displays aSyn-GFP expression at wt levels, only in neurons and with the same distribution as wt aSyn. Moreover, the fusion-protein localizes correctly to synaptic vesicle and participates in the synaptic vesicle cycle. Importantly, aSyn-GFP is incorporated in Lewy-like pathology seeded by exposure to PFFs. We anticipate that this new tool will allow for further studies aimed at better understanding aSyn physiology and pathobiology.

Snca-GFP knock-in mice express wildtype levels of aSyn-GFP fusion protein
We generated a novel mouse line that genetically encodes fluorescent aSyn by using homologous recombination to insert the cDNA for enhanced-GFP into the Snca locus of murine embryonic stem cells ( Figure 1A; see also Methods). The construct was targeted to the 3' end of exon 6 while keeping the surrounding endogenous regulatory 7 elements intact. The modified Snca-GFP gene was predicted to transcribe a fusion protein consisting of wild type (wt) murine aSyn with a C-terminal GFP tag. Embryonic stem cells containing the recombined construct following electroporation were confirmed by Southern blot analysis ( Figure 1A, right panel) and a validated clone (1B6) was implanted in C67/B6 blastocysts to yield founder lines. Two founder mouse lines were derived and offspring with normal karyotypes were further expanded by breeding out onto a C57/Bl6J background. PCR genotyping with primers targeting exon-intron boundaries revealed mice with either wt, heterozygous (Snca wt/GFP ), or homozygous (Snca GFP/GFP ) knock-in genotypes ( Figure 1B). Both knock-in genotypes were fertile and did not show detectable differences in lifespan nor any overt behavioral defects up to two years of age.
Upon illumination with blue light, brains and spinal cords from Snca wt/GFP and Snca GFP/GFP mice fluoresced brightly, indicating expression of the aSyn-GFP fusion protein in tissues where aSyn is normally abundant ( Figure 1C). Quantification of aSyn mRNA by RT-PCR showed that total transcript levels did not differ between KI and wt mice ( Figure 1D). We further confirmed that this represented the expression of an intact aSyn-GFP fusion protein by subjecting brain homogenates derived from wt and KI mice to immunoblot analysis. Probing with antibodies against aSyn and GFP showed a 45-kDa product corresponding to the predicted molecular weight of aSyn-GFP in Snca GFP/GFP and Snca wt/GFP littermates that was absent from wt controls ( Figure 1E). No significant cleavage products were detected by either antibody. Anti-aSyn antibodies appeared to showed stronger reactivity for aSyn-GFP relative to the untagged form by immunoblot (Figure 1 E and Extended Figure 1). Nonetheless, Snca wt/GFP mice expressed both aSyn-GFP and untagged aSyn at ~50% of the amount of aSyn seen in Snca GFP/GFP and wt 8 mice, respectively ( Figure 1E, lower panel). These results indicate that Snca-GFP mice express physiological levels of an aSyn-GFP fusion protein with minimal disruption of endogenous mouse aSyn mRNA and protein levels.

Regional and subcellular localization of aSyn-GFP in Snca-GFP mice is identical to wt animals
Since the endogenous promoter and regulatory elements remain unmodified in Snca-GFP mice, we predicted that the regional and subcellular distribution of aSyn-GFP would be comparable between wt and both KI genotypes. Examination of native GFP fluorescence in 40 µm-thick brain and spinal cord sections revealed abundant aSyn-GFP expression particularly in the hippocampus, substantia nigra, striatum, cerebral cortex, globus pallidus, thalamus and olfactory bulb (Figure 2A), mirroring the endogenous pattern previously reported for C57BL/6 mice (Taguchi et al., 2016). Detection by immunofluorescence also showed no obvious differences in regional aSyn distribution between brain sections from Snca wt/wt , Snca wt/GFP and Snca GFP/GFP mice ( Figure 2B). In addition, GFP-immunoreactivity overlapped completely with that of aSyn in Snca wt/GFP mice, further indicating the intact tagged protein is normally expressed nor does it perturbate the distribution of wt aSyn ( Figure 2C).
Co-immunostaining of GFP with markers for different CNS cell types showed that aSyn-GFP is expressed by neurons but not detectable in astrocytes, oligodendrocytes or microglial cells ( Figure 3A). Within neurons, aSyn-GFP co-localized with glutamate vesicular transporter (VGLUT; Figure 3B), a presynaptic marker, consistent with the known enrichment of aSyn in presynaptic vesicles in excitatory neurons (Burre, 2015;9 Maroteaux et al., 1988). aSyn-GFP was also detectable in brain areas containing nonglutamatergic neurons (e.g., striatum and olfactory bulb), where it co-localized with glutamate decarboxylase (GAD) positive neurons ( Figure 3B and data not shown).
Together, these results suggest that the in vivo distribution of the aSyn-GFP fusion protein closely matches that of wt aSyn at the regional, cellular, and subcellular levels.

aSyn-GFP is enriched in presynaptic vesicles in Snca-GFP primary neurons
To further determine the utility of these KI mice for studying aSyn pathobiology, we compared the expression levels and subcellular localization of aSyn-GFP in primary neurons derived from Snca wt/GFP , Snca GPF/GFP and wt mice. Hippocampal and cortical neurons ( Figure 4 and data not shown) prepared from postnatal day 0 Snca wt/GFP and Snca GPF/GFP mice developed normally in culture, with aSyn-GFP detectable by DIV 7.
aSyn-GFP was mainly enriched in vesicles, similar to what is seen in Snca-GFP brains ( Figure 4A). Both wt aSyn and aSyn-GFP expression in cultured neurons increased with age before plateauing at DIV 10, indicating that the addition of the GFP-tag does not alter the developmentally regulated expression of aSyn in neurons (data not shown). In mature cultures, aSyn-GFP colocalized strongly with Synapsin 1, a marker of synaptic vesicles ( Figure 4A). In addition, aSyn and aSyn-GFP strongly overlap in Snca-GFP wt/GFP neurons, which express equal quantities of both ( Figure 4B). Therefore, neurons prepared from Snca-GFP mice exhibit the expected synaptic distribution of aSyn and Synapsin 1.
Immunoblot analysis of hippocampal culture lysates with aSyn and GFP antibodies confirmed the expression of a single major species consistent with intact aSyn-GFP at the expected ratios with respect to the wt protein in each genotype ( Figure 4C). Taken together, aSyn-GFP maintains normal subcellular expression and distribution without disrupting the morphology of synaptic vesicles.

aSyn-GFP participates in the synaptic vesicle cycle with kinetics similar to wt aSyn
To further establish that aSyn-GFP has similar functional properties to wt aSyn, we examined its ability to participate in synaptic vesicle cycling in primary hippocampal neurons. In wt neurons, aSyn is dispersed following stimulation with 90 mM KCl and partially repopulates into vesicles after stimulus removal (Fortin et al., 2005). We therefore performed live imaging on DIV18-22 Snca w/tGFP hippocampal neurons to monitor the redistribution of aSyn-GFP during stimulation and subsequent recovery. Addition of KCl induced a marked decrease in aSyn-GFP intensity in individual vesicles within 2 minutes of treatment ( Figure 5A and 5B), in agreement with the dynamics previously reported for wt aSyn (Fortin et al., 2005). Neurons were also fixed and immunostained for Synapsin 1, which is known to disperse following stimulation and redistribute back to synaptic vesicles shortly after the stimulus end (Chi et al., 2001). Unlike Synapsin 1, aSyn-GFP did not fully re-establish its vesicular localization after a 15-minute recovery period following KCl treatment Figure 5C). These results further indicate that aSyn-GFP is functionally similar to its wt counterpart.

aSyn-GFP is detectable in multiple peripheral cell types
Since expression of aSyn-GFP in Snca-GFP mice remains under the control of the endogenous promoter and regulatory elements, we predicted that its distribution in peripheral organs would also parallel that of the wt protein. We therefore surveyed aSyn-11 GFP expression in a variety of organs across multiple anatomical regions. As previously reported for wt aSyn (Barbour et al., 2008), GFP fluorescence was prominent in blood (primarily erythrocytes; Figure 6A) and bone marrow (data not shown). Endogenous GFP fluorescence in blood was also detectable in Snca GFP/GFP mice by spectrophotometry ( Figure 6B) and confirmed by immunoblotting in lysates prepared from sedimented red blood cells ( Figure 6C). We also detected GFP fluorescence in unfixed colon samples ( Figure 6D, left panel) in which aSyn-GFP could be found in neurons of the myenteric plexus. Although the native GFP signal was lost after fixation, expression of the fusion protein was confirmed in this tissue using an anti-GFP antibody ( Figure 6D, right panels).
In contrast, GFP fluorescence in most other tested tissues was indistinguishable from wt mice suggesting levels below our detection limit, consistent with previous studies.

GFP-tag partially inhibits but does not abolish aSyn fibril assembly
The normal distribution and functioning of aSyn-GFP in Snca-GFP mice led us to examine its utility for investigating the pathobiology of aSyn, specifically its formation into amyloid fibrils that accumulate within intracellular Lewy pathology found in PD and DLB (Arima et al., 1998;Takahashi and Wakabayashi, 2001). Since previous studies showed that the presence of a GFP tag can alter the rate of aSyn aggregation (Afitska et al., 2017), we tested whether recombinant aSyn-GFP can self-assemble into fibrils under conditions where wt aSyn readily polymerizes. Independent reactions containing either recombinant wt aSyn or aSyn-GFP monomer were incubated with agitation as previously described (Luk et al., 2016). Within 6h of the start of the reaction, the majority of wt aSyn had converted to insoluble species which accounted for nearly all aSyn by 8h (Figures 12 7A-C). In parallel reactions, aSyn-GFP also accumulated in the insoluble fraction over time, but with significantly delayed kinetics ( Figure 7A-C). When equimolar concentrations of untagged aSyn and aSyn-GFP were combined, both forms aggregated at a similar rate that fell between that of each individual protein ( Figure 7C). Inspection of the products from these reactions by electron microscopy ( Figure 7D), revealed the presence of filamentous structures compatible with those previously reported for aSyn (Luk et al., 2009). Of note, aSyn-GFP fibrils assembled in vitro are able to induce aSyn pathology in wt and Snca-GFP neurons ( (Karpowicz et al., 2017) and data not shown). Taken together, aSyn-GFP is fibril assembly-competent, albeit with slower kinetics, which may be mitigated in the presence of untagged aSyn monomer.

Snca-GFP neurons
We and others have previously demonstrated that aSyn fibrils internalized by neurons can template the conversion of endogenously expressed aSyn into fibrillar forms that accumulate as Lewy-like inclusions (Volpicelli-Daley et al., 2011). We therefore determined whether primary hippocampal neurons from Snca-GFP mice are permissive to such pathological seeding following exposure to aSyn PFFs and incorporated aSyn-GFP into insoluble intraneuronal inclusions. Neurons from Snca wt/GFP mice developed inclusions resembling Lewy neurites and Lewy bodies when exposed to mouse wt PFFs, although the level of pathology, as measured by pSer129 aSyn (pSyn) immunostaining, was reduced relative to similarly treated wt neurons ( Figure 8 A and B). In agreement with our in vitro data showing that the rate of aggregation is reduced when only aSyn-GFP is 13 present, pathology formation at the same time point was further reduced in Snca GFP/GFP neurons. Nevertheless, pSyn was still detectable in a small proportion (~0.005% of wt) of PFF-exposed Snca GFP/GFP neurons ( Figures 8A and 8B).
In both Snca wt/GFP and Snca GFP/GFP neurons, co-labeling with antibodies to pSyn and GFP revealed near-complete co-localization within inclusions, indicating that aSyn-GFP was uniformly incorporated ( Figure 8D and data not shown). Biochemical analysis of Triton-X100 insoluble proteins from these cultures also confirmed that a majority of aSyn-GFP shifted to this fraction after PFF treatment ( Figure 8C).
We further determined whether aSyn-GFP can also undergo pathological conversion in vivo by using intracranial injection to target PFFs into the mouse brain, a model that allows the induction and propagation of aSyn pathology in presence of wt levels of this protein. For this, we selected the hippocampus due to previous reports (Luna et al., 2018;Nouraei et al., 2018)  develop pathology during normal aging (data not shown) marking a notable difference with previous models overexpressing GFP-tagged aSyn (Hansen et al., 2013).

Discussion
Clinical and experimental evidence implicate aSyn in multiple neurodegenerative disorders. However, neither the normal function and regulation of aSyn, nor its role in disease initiation and progression, are fully understood. Given the dynamic nature of these biological processes, tools that enable the direct visualization of aSyn would facilitate efforts to address these fundamental questions. Moreover, the majority of individuals with synucleinopathy carry neither aSyn mutations nor overexpress aSyn to any measureable extent. To this end, we generated a aSyn-GFP knock-in (KI) mouse line in which endogenous aSyn protein is fused to GFP via its carboxy-terminal with the goal of combining the utility of a genetic-encoded fluorescent tag while preserving the protein's natural distribution throughout the body.
Our data here demonstrate that both heterozygous (Snca wt/GFP ) and homozygous (Snca GFP/GFP ) mice express aSyn-GFP in a pattern that is indistinguishable from aSyn protein in wt animals across multiple tissues including, but not limited to, the central and enteric nervous systems, erythrocytes, and bone marrow. Within the brain, where aSyn is highly enriched, total aSyn mRNA and protein levels were comparable between wt and KI animals while expression levels of aSyn-GFP were directly proportional to gene dosage. In contrast to some previous reports, aSyn-GFP appeared to be expressed primarily in its intact form and minimal levels of degraded products were detected (McLean et al., 2001). We speculate that expression of aSyn-GFP at endogenous levels and the inclusion of the short linker between aSyn and GFP may contribute to this stability.
When examined in vivo or in cultured neurons derived from KI mice, aSyn-GFP is detectable in all neurons and is correctly localized to a subset of synaptic vesicles, while the complete co-localization between native and GFP-tagged aSyn in Snca wt/GFP mice suggests that aSyn-GFP does not perturb the expression or localization of its wt counterpart. Indeed, the addition of the GFP-tag appears to have a minimal effect on a complex aspect of aSyn function (i.e., participation in synaptic vesicle cycling) as both wt and tagged aSyn disperse similarly after synaptic stimulation, in agreement with previous studies (Fortin et al., 2005;Unni et al., 2010).
The above characteristics distinguish this KI line from previously reported mammalian models that employ a genetically encoded GFP tag to label aSyn. In these, the tagged sequence corresponds to wt human aSyn with expression regulated by either a non-Snca promoter (e.g. PDGFβ) (Rockenstein et al., 2005) or downstream of the mouse Snca promoter by means of a bacterial artificial chromosome (Hansen et al., 2013). Although robust neuronal expression was achieved in both examples, the distribution and levels of tagged aSyn did not precisely match that of aSyn in the nontransgenic host and aSyn was also ectopically expressed in additional cell types and regions. Interestingly, GFP-tagged aSyn in these mouse lines also accumulate as lysosome-associated inclusions or undergo phosphorylation at Ser129, a marker of Lewy body and Lewy neurite pathology in human synucleinopathies, with aging. A possible explanation for such differences is that the previously described lines were selected based on high expression levels of the transgene, among other criteria. Total aSyn levels in these mice may have been further amplified by maintaining these mice on a wildtype genetic background without deletion of the endogenous Snca locus. In contrast, the localization and levels of aSyn-GFP in Snca wt/GFP and Snca GFP/GFP mice matched that of wt mice and we did not observe any redistribution or modification of aSyn-GFP in mice up to 2 years of age. We believe these features make aSyn-GFP KI mice particularly amendable for investigating aSyn trafficking and action, a crucial but understudied area of aSyn biology, especially given that these processes are highly sensitive to aSyn levels (Eguchi et al., 2017;Scott and Roy, 2012).
An added advantage is that aSyn-GFP is normally distributed throughout peripheral organs such as neurons in the gastro-intestinal tract and red blood cells in this line. As we demonstrate, this expression also permits rapid quantification of aSyn levels without the need for additional sample processing (e.g. immunostaining). It is anticipated that this will further enable the validation of endogenous aSyn expression in other peripheral tissues, especially those where aSyn is in low abundance, and where detection using immunohistochemistry alone has provided equivocal results. Primary cells derived from aSyn-GFP KI mice are also a resource for investigating the aSyn biology at the cellular level.
In addition to physiological functioning, our work here demonstrates that Snca wt/GFP and Snca GFP/GFP neurons can also serve as permissive cellular host for pathological seeding by misfolded aSyn species. Specifically, recombinant aSyn PFFs induced GFP-positive Lewy-like pathology in cultured hippocampal neurons when introduced into the culture media or in multiple CNS regions when PFFs are stereotaxically injected into either dorsal striatum or hippocampus. Importantly, fibril-induced pathology in Snca wt/GFP and Snca GFP/GFP was distributed in the same brain regions as in wt mice, albeit with different densities, suggesting a similar spreading process within neuroanatomical pathways. The intact aSyn-GFP moiety represented the major species in these intraneuronal inclusions, confirming that this pathology is formed predominantly by aSyn-GFP derived from the neuronal pool and is consistent with previous studies showing that neurons overexpressing aSyn-GFP support fibril-induced pathology formation (Osterberg et al., 2015;Volpicelli-Daley et al., 2014). Interestingly, the proportion of untagged aSyn and aSyn-GFP found in the intracellular inclusions in PFF-treated Snca wt/GFP neurons were similar, providing further support that the tagged protein is converted under a similar process.
Although our data clearly shows that aSyn-GFP can polymerize into fibrils and can be recruited into Lewy-like pathology in neurons, the kinetics of this process is altered by the presence of the GFP tag compared to wt untagged aSyn both in vitro and in vivo. In summary, we have generated a novel in vivo resource for studying multiple aspects of aSyn function under physiological and disease-like conditions without genetic overexpression. Snca-GFP mice provide the opportunity to concomitantly track and measure soluble and pathological forms of the protein across relevant tissues. We anticipate that future studies leveraging these animals (e.g., by crossbreeding with other genetic models of disease) should provide additional insights into aSyn biology.

Animals
All housing, breeding, and procedures were performed according to the NIH Guide for the Care and Use of Experimental Animals and approved by the University of Pennsylvania Institutional Animal Care and Use Committee. Animals were anesthetized with a mix of Ketamine: 100 mg/kg; Xylazine: 10 mg/kg; Acepromazine: 0.5 mg/kg before performing transcardiac perfusion with PBS + heparin (2 USP/ml). The Snca-GFP KI line was created by homologous recombination: a synthetic mouse Snca exon 6 with a short linker (the primary sequence around the linker being …EPEA-KL-MVSKG…) was inserted directly after the last amino acid of the mouse Snca coding sequence, followed by the enhanced GFP coding sequence and the remainder of the SNCA 3'UTR ( Figure 1A). A synthetic DNA construct (Blueheron Inc.) consisting of ~200 nt of the genomic Snca sequences upstream of exon 6 and all of exon 6 (with the above linker and eGFP sequence inserted directly after the C-terminus of Snca) was cloned directly downstream of the floxed Neo cassette of PL452 (NCI, Frederick). A ~500 nt long Snca upstream arm of homology was added upstream of the Neo cassette. This construct was introduced by BAC recombineering into pL253 (NCI, Frederick) harboring ~12 kB of the genomic SNCA region containing at its center SNCA exon 6.
The resulting targeting vector contained a 14183 nt long Snca allele with integrated floxed Neo cassette as well as Snca exon 6 containing KL -eGFP downstream of the Snca protein. The targeting vector was linearized with NotI and introduced into V6.5 ES cells by electroporation. After geneticin selection ES clones were screened by Southern blotting (HindIII digestion) and 5 positive clones were found and further validated by a second round of Southern blotting (PvuII digestion) using a different probe (see Figure 1 A). The positive clones generated a wt ~12 kB and a mutant ~9kB PvuII fragment.
Positive clones were subjected to chromosome counting. Clone 2B9, which had the correct number of chromosomes, was injected into C57/B6 mouse blastocysts.
Heterozygous and homozygous mice were obtained and confirmed by PCR using primers encompassing the fusion region (Exon 6) that amplify a longer fragment when the GFP sequence is present (See Figure 1A

Reagents and Chemicals
All reagents were purchased from Fisher and all chemicals were purchased from Sigma unless otherwise indicated

Recombinant aSyn monomers and PFFs
Recombinant mouse aSyn and aSyn-GFP were produced as previously described (Karpowicz et al., 2017;Luk et al., 2012) . In brief, plasmids encoding the 2 proteins were introduced in E. coli using heat shock and proteins were purified following lysis using size exclusion and anion exchange chromatography. Purified protein (monomer) was concentrated to 5 mg/ml for aSyn wt and 15 mg/ml for aSyn GFP (360 uM) and kept frozen until use. Recombinant PFFs (mouse wt aSyn) were produced by shaking 500 ul of recombinant aSyn monomer (5 mg/ml, 360 uM) at 37 C for 7 days. Fibril formation was confirmed by sedimentation assay (see specific section). Right before their use, PFFs are diluted in PBS and sonicated for 10 cycles (1 sec ON, 30 sec OFF, high intensity) in a bath sonicator at 10°C (BioRuptor; Diagenode).

Sedimentation assay
Monomers were thawed and ultracentrifuged at 100,000 g for 30 minutes at RT. They were then diluted to 2.5 (wt aSyn, 180 uM) and 7.5 mg/ml (aSynGFP, 180 uM) in PBS.
100 ul of asyn, aSyn GFP or aSyn + aSyn GFP (1:1) were aliquoted in Eppendorf tubes and shaken at 1,000 rpm at 37°C for 24h. 6 ul of each reaction was collected at the indicated time points, diluted in 60 ul of PBS and layered on a 25% sucrose cushion before undergoing ultracentrifugation (30 mins at 100,000 x g). Supernatant and pellet fractions were separated. Pellets were resuspended in 120 ul of 12.5% sucrose. 25 ul of 5X SDS sample buffer were added to each tube and 30 ul was loaded on 15% acrylamide gels for SDS PAGE. Separated proteins were then stained with Coomassie Blue ON and destained with 10% isopropanol, 10 % acetic acid and water before image acquisition with an infrared scanner (LiCor Odyssey).

Sequential extraction
Neurons were washed twice with PBS and lysed in TBS (50 mM TRIS, 150 mM NaCl, pH=7.6) + 1% Triton X-100. Lysates were sonicated for 1 second ON, 30 seconds OFF, medium intensity in a bath sonicator at 10 C (BioRuptor; Diagenode) and then rotated at 4 C for 20 minutes. Lysates were then spun at 100,000 x g for 30 mins at 4°C, supernatants were removed and labelled as TX-100 soluble fraction. The pellet was washed once with 1% TX-100 TBS, sonicated and ultracentrifuged. Supernatants were discarded and pellets dissolved in 1/10 volume of TBS, 2% SDS, sonicated and 22 ultracentrifuged. Supernatants were collected as SDS soluble fraction and pellets were discarded. Protein content was measured by BCA in the TX-100 soluble fraction. Equal amount of total proteins from TX-100 soluble fractions were separated by SDS-PAGE (4-20% gradient gel). An equal volume of the SDS soluble fraction was loaded alongside. Proteins were detected by western blot with the indicated antibodies.

Immunofluorescence
Primary neurons: Neurons were fixed at the indicated DIV with warm 4% paraformaldehyde+ 4% sucrose for 15 minutes at RT. Cells were permeabilized and non-specific binding sites blocked using PBS containing 0.1% TX-100, 3% BSA, 3% FBS for 20 minutes. Primary antibodies were added to cells for 1 h, followed by 3 washes with PBS and 1 h incubation with the appropriate secondary antibody (1:1 K). Cells were washed 3 times with PBS, once in water and mounted using Fluoromount-G. 40 um brain sections and intestinal whole mounts: sections were permeabilized and blocked in PBS containing 10% FBS, 3% BSA, 0.5% TX-100 for 1 hour at RT, incubated with primary antibodies ON at RT, washed 3 times with PBS, incubated with secondary antibodies for 2 hours at RT, washed and mounted using Fluoromount-G (brain sections) or 1:1 PBS/glycerol (intestine). 6 um sections: sections were produced and treated as described in the immuonohystochemistry section with the modification that after the ON incubation with primary antibodies, sections were incubated with a fluorescently labelled secondary antibody for 2 hours at RT and mounted using Flouromount-G.
Images were captured on a Nikon Ds-Qi1Mc digital camera attached to a Nikon Eclipse Ni microscope (6 um sections), a Leica Confocal SP8 for colocalization studies (primary neurons and 40 um sections) or using InCell Analyzer 2200 (GE Healthcare) when using 96 well plates). Analysis was performed using Fiji or Developer software (GE Healthcare, 96 well plates).
Immunocytochemistry PBS-perfused mouse brains were post fixed in ethanol (70% in 150 mM NaCl) ON at 4°C, cut in 3 mm slabs, and embedded in paraffin. Tissue was then sectioned at 6 micrometers using a microtome and applied on glass slides. Before staining, sections were de-paraffinized and rehydrated (xylene, 95%, 90% and 75% ethanol). Sections were blocked in TBS containing 3% FBS and 2% BSA for 1 hour at RT, incubated with primary antibodies at 4 C ON, with biotinylated secondary antibodies for 1 hour at RT (Vector Laboratories), horse radish peroxidase conjugated streptavidin for 1 hour at RT (Vector Laboratories), and signal was revealed using DAB peroxidase substrate products (dark brown, Vector Laboratiories). Sections were counterstained with hematoxylin for 1 min and mounted using Cytoseal Mounting Media. Images were captured on a Nikon Ds-Qi1Mc digital camera attached to a Nikon Eclipse Ni microscope or using Lamina Scanner (PerkinElmer; 20X objective).

Synaptic vesicle cycling
Hippocampal neurons were plated on IBIDI dishes and kept in culture for 18-21 days.
Cells were then incubated in Krebs Ringer HEPES buffer (5 mM KCl, 140 mM NaCl, 10 mM HEPES, 10 mM Glucose, 2.6 mM CaCl2, 1.3 mM MgCl2) for 15 minutes before starting the imaging sessions. Images were acquired with a Leica microscope and a 40X air objective. Cells were imaged (every 3-5 seconds) in KRH solution for 2-3 minutes to establish a baseline signal. The media was then switched to high potassium (HK; 90 mM KCl, 55 mM NaCl, 10 mM HEPES, 10 mM Glucose, 2.6 mM CaCl2, 1.3 mM MgCl2) or KRH solution for 2 minutes. Individual vesicles were fragmented using ImageJ/FIJI and intensities before and after stimulation were determined as ratios after subtracting background signal and adjusting for image drift (Lazarenko et al., 2018).
For fixed samples, cells on coverslips were washed and incubated in KHR for 15 minutes at RT and then either fixed immediately before treatment, incubated with HK for 2 mins before fixation (HK), or washed for an additional 15 mins using HRK before fixation (HK+15' recovery). Cells were then processed for immunostaining as described above.

Western Blot
Cells were lysed in lyses buffer (0.5% Triton X-100, 0.5% deoxycholic acid, 10 mM TRIS, 100 mM NaCl, pH=8.0) with phosphatase and protease inhibitors. Nuclei and debris were removed by centrifugation (5 mins at 1,000 x g). Total protein content was determined by BCA and equal amounts of total protein were separated on 4-20% gradient gels. Proteins were then transferred to 0.22 um nitrocellulose membranes (1 hour at 100 V at 4 C). Membranes were blocked in 7.5 % BSA and incubated with the 25 indicated primary antibodies ON followed by appropriate secondary antibodies (LiCor) for 1 hour at RT. Image acquisition was performed using a Licor Scanner and image analysis performed using ImageJ/FIJI (NIH).

RT-PCR
Mice were perfused, brain removed in conditions that minimize RNA degradation and half hemispheres were frozen for further processing. Frozen tissue was thawed and immediately homogenized in 2 ml of RTL buffer + 2mM DTT (Qiagen). mRNA was extracted from 300 ul of homogenate using the RNeasy Kit (Qiagen) according to manufacturer's instructions. 200-800 ug of mRNA were converted to single strand cDNA with SuperScript™ III First-Strand Synthesis System (Thermo Fisher) using random hexamers and the protocol detailed by the vendor. Brain derived cDNA and primers targeting mouse a-Syn (see table) were used in Syber Green (Thermo Fisher) Real Time PCR reactions monitored by a 7500 Fast Real Time PCR system (Applied Biosystems). Actin and SNAP 25 were used as internal controls (see table). Data is expressed as fold change over the wild type genotype.

Measurements of GFP signal in mouse blood
Total blood was collected in EDTA containing tubes by cardiac puncture in deep terminal anesthesia and right before transcardial perfusion (see specific section). 25 ul of blood were lysed by addition of 225 ul of lysis buffer and 5 ul of lysate were diluted in 190 ul of water and analyzed for fluorescence using a spectrophotometer (excitation: 488 nm; emission: 530 nm).

Experimental designs and Statistical analysis
27 Details for each experiment and statistical analysis are described in the Figure Legends. Statistical analysis was performed using GraphPrism (v7) or GraphPrism (v8 for figure   7C).             Snca wt/ wt Snca wt/ GFP Snca GFP/ GFP