Carboxy-terminal truncations of mouse alpha-synuclein alter aggregation and prion-like seeding

Abstract

α-synuclein (αsyn) forms pathologic inclusions in several neurodegenerative diseases termed synucleinopathies. The inclusions are comprised of αsyn fibrils harboring prion-like properties. Prion-like activity of αsyn has been studied by intra-cerebral injection of fibrils into mice, where the presence of a species barrier requires the use of mouse αsyn. Post-translational modifications to αsyn such as carboxy-terminal truncation occur in synucleinopathies, and their implications for prion-like aggregation and seeding are under investigation. Herein, carboxy-truncated forms of αsyn found in human disease are recapitulated in mouse αsyn to study their seeding activity in vitro, in HEK293T cells, in neuronal-glial culture, and in non-transgenic mice. The results show that carboxy-truncation of mouse αsyn accelerates aggregation of αsyn but alters prion-like seeding of inclusion formation.

Introduction

Parkinson’s disease (PD) and Lewy body dementia (LBD) are neurodegenerative disorders characterized by neuronal inclusions termed Lewy bodies (LBs) and Lewy neurites, which are comprised of misfolded αsyn assembled into pathologic fibrils (1). These aggregated αsyn fibrils harbor prion-like activity such that they can induce endogenous monomeric αsyn to misfold, catalyzing more fibril formation that is postulated to spread inter-neuronally (2). Pre-formed αsyn fibrils (PFFs) are sufficient to induce toxicity and inclusion formation in non-transgenic (NTG) mice, suggesting that the formation of these toxic fibrils is sufficient to start a pathologic cascade resulting in progressive disease (3). Intra-cerebral injection of PFFs has been a useful model to investigate mechanisms of synucleinopathy progression and has potential for therapeutic developments (2), however similar to prions a species barrier exists whereby PFFs comprised of mouse αsyn are much more potent than human αsyn at inducing extensive inclusions to form in NTG mice (4).

One mechanism by which αsyn may gain toxic aggregation properties is through post-translational modifications common in disease including phosphorylation, ubiquitination, and truncation (5, 6). Carboxy-truncation (C-truncation) of αsyn is one modification that has been shown in vitro and in cultured cells to increase the propensity of monomeric αsyn to spontaneously aggregate and also to modulate the prion-like seeding activity of the fibrils (7–9). Recent characterization of αsyn fibrils through the use of cryo-electron microscopy and other techniques suggests that C-terminus truncation modifications can greatly alter αsyn fibril structure (9–11); consequent alterations in prion-like seeding as a result of these modifications may be important in explaining strain-like diversity in fibril structure and seeding capacity isolated from LBs (12, 13). Additionally, recent works have suggested that PFFs when introduced to healthy neurons are quickly trafficked for lysosomal processing where extensive truncation of exposed C-terminal regions on the PFFs can readily occur (2, 14–16); this indicates that initial seeding events in a neuron likely involve truncated αsyn fibrils. Indeed, various truncated forms of human αsyn have been recently investigated and it has been found that seeding activity can increase or decrease depending on the specific truncation (7, 8, 15). Herein, a subset of the most common C-truncated forms of αsyn (7) are investigated with mouse αsyn for compatibility with NTG mouse-based models of prion-like seeding to further characterize how C-truncation affects induction of pathology.

Materials and Methods

Antibodies

Mouse monoclonal antibody 81A (RRID: AB_2819037) is mainly specific for phosphorylated Ser129 (pSer129) αsyn and is a common diagnostic marker of pathologic αsyn (17, 18); the antibody was used at a 1:3000 dilution for immunohistochemistry. Antibody 94-3A10 (RRID: AB_2819044) is a mouse monoclonal antibody specific for an epitope located at the last 10 C-terminal amino acid of αsyn (residues 130-140 αsyn) (19, 20); the antibody was used at a 1:10,000 dilution for immunohistochemistry and 1:1000 for western blotting. SNL-4 (RRID: AB_2819043) is a rabbit polyclonal antibody raised against residues 2-12 of αsyn (21); the antibody was used at a 1:1000 dilution for western blotting.

Expression and purification of recombinant αsyn proteins

QuikChange site-directed mutagenesis (Agilent Technologies, Santa Clara, CA) using mutant-specific oligonucleotides was used to generate various C-terminally truncated forms of mouse αsyn (residues 1-115, 1-119, 1-122, 1-124, 1-125, 1-129, 1-133, and 1-135) in cDNA encoding full-length (FL) mouse αsyn in both the pRK172 and pcDNA3.1 (+) plasmid as previously described (22). The pRK172 DNA construct expressing N-terminal truncated 21-140 mouse α-syn (with a Met codon added before amino acid 21) was generated by PCR amplification and cloning in pRK172. All recombinant forms of αsyn were expressed in E.coli BL21 (DE3) and purified as previously described (7). All recombinant proteins were diluted in pH 7.4 sterile phosphate buffered saline (PBS), and concentrations were determined using the bicinchoninic acid assay (Pierce, Waltham, MA).

In vitro aggregation of C-truncated mouse αsyn and preparation of fibrils

For in vitro aggregation comparisons, FL and C-truncated mouse αsyn proteins were diluted to 100 μM in sterile PBS (Invitrogen) and assembled into amyloid fibrils with continuous shaking at 1050 rpm, 37°C, for 0, 24, 48, and 96 hours with 4 replicates per form of αsyn protein at each time point. Amyloid formation for each tube (n = 4) was assessed by determining the fraction of insoluble αsyn at each timepoint (40 μL of solution) as previously described (7). Insoluble αsyn was measured by centrifugation at 100,000 g for 30 minutes in PBS to produce a soluble supernatant and insoluble pellet fraction; SDS-PAGE was conducted to determine the fraction of insoluble αsyn as described previously (7).

For seeding experiments, all forms of mouse αsyn were individually assembled into pre-formed mouse αsyn fibrils (mPFFs) by incubation at 37°C at 5 mg/ml in sterile PBS with continuous shaking at 1,050 rpm. αsyn fibril formation was validated with K114 fluorometry as previously described (7). When combinations of 1-135 with C-truncated forms of αsyn were co-fibrillized, a 1:1 molar ratio was used at a 5 mg/ml total concentration. PFFs were diluted in sterile PBS and fragmented into an array of shortened fibrils by mild water bath sonication for one hour prior to seeding in cell culture or intra-hippocampal injection (23).

HEK293T cell culture and transfection

HEK293T cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Invitrogen, Carlsbad, CA) containing 2 mM L-glutamine, 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin, at 37°C and 5% CO2. Cells were plated into 4 cm² wells, and allowed to reach ~30% confluency; cells were transfected using a modified calcium phosphate protocol (7). For each of 3 replicate wells per construct, 1.5 μg of pcDNA3.1 vector expressing FL or one of 8 forms of C-truncated mouse αsyn was used for transfection. One hour after transfection, mPFFs comprised of m21-140 αsyn or one of 8 forms of C-truncated mouse αsyn were added to 1 μM in each well, with the concentration being based on that of the monomeric subunits. At 16 hours post transfection, cells were washed with PBS and media was changed to DMEM culture media containing 3% FBS. Cells were harvested for biochemical fractionation at a final time point of 64 hours post transfection.

Primary neuronal-glial culture (PNC) preparation and experimentation

Embryonic day 16-18 (E16-18) B6C3F1 background mice (Charles River) were used to prepare primary mixed neuronal-glial cultures from cerebral cortices and maintained for 16 days total as previously described (7, 16). Cultures prepared using this method are usually 20% neuronal and 80% glial in composition (16). To study seeded aggregation in these cultures, mPFFs comprised of C-truncated mouse αsyn were added to a concentration of 1.5 μM at culture day 6 and maintained with half-volume media changes every 4 days until harvest for biochemical characterization at culture day 16 as previously described (7).

HEK293T and PNC biochemical fractionation and western blot analysis

HEK293T cells and PNC cultures (n = 3 for all well replicates) were lysed in 200 μL/well detergent extraction buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 20 mM NaF) supplemented with protease inhibitors (1 mM phenylmethylsulfonyl and 1 mg/ml each of pepstatin, leupeptin, N-tosyl-L-phenylalanyl chloromethyl ketone, N-tosyl-lysine chloromethyl ketone and soybean trypsin inhibitor) as previously described (7). Lysate was separated into Triton X-100 (triton) soluble and insoluble fractions; equal volumes were loaded onto 15% polyacrylamide gels, resolved by SDS-PAGE, and transferred onto 0.2 μm pore size nitrocellulose membranes (Bio-Rad, Hercules, CA), in carbonate transfer buffer (10 mM NaHCO₃, 3 mM Na₂CO₃, pH 9.9) (24). Membranes were blocked in 5% dry milk/Tris buffered saline (TBS) and incubated overnight at 4°C with primary antibody (SNL4 or 94-3A10) and diluted in block solution. After washing in TBS, membranes were incubated in goat anti-secondary antibody conjugated to horseradish peroxidase (Jackson Immuno Research Labs, Westgrove, PA) diluted in 5% dry milk/TBS for 1 hour; detection of αsyn was accomplished using Western Lightning-Plus ECL reagents (PerkinElmer, Waltham, MA) followed by chemiluminescence imaging (PXi, Syngene, Frederick, MD). Densitometry was performed using ImageJ software to quantify the fraction of insoluble αsyn.

Mice

All animal experimental procedures were performed in accordance to University of Florida Institutional Animal Care and Use Committee regulatory policies following approval. Mice were housed in a stable environment with a 12-hour light/dark cycle and access to food and water ad libitum. In total, 27 NTG mice of the B6C3F1 background (Charles River) were utilized for surgical experimentation.

Intra-hippocampal injection of mPFFs

For comparison of prion-like seeding properties between FL and C-truncated forms of αsyn, 3 cohorts of 8 mice were stereotactically injected unilaterally into the hippocampus (coordinates from Bregma: anterior/posterior −2.2 mm, lateral ±1.6 mm, dorsal/ventral −1.2 mm) at ~2 months of age as previously described (23). For injection, 2 μL of solution (sterile PBS) containing 4 μg of FL mPFFs or the molar equivalent of mPFFs comprised of either 1-125 or 1-129 C-truncated mouse αsyn were utilized. An additional 3 mice were injected in the same location with 2 uL of vehicle (sterile PBS) as negative controls. At 3 months post-surgery, animals were sacrificed for histologic analysis.

Tissue preparation and immunohistochemistry

Mice were euthanized with CO2, perfused with a heparin/PBS solution, fixed in 70% ethanol/150 mM NaCl and sectioned as previously described (25). For immunohistochemical analysis, sections were stained utilizing established methods (25, 26). Antigen retrieval was performed utilizing target retrieval solution (Agilent, Santa Clara, CA) in a steam bath for 1 hour. All antibodies were incubated with tissue overnight at 4°C overnight in block solution (2% FBS in 0.1 M Tris pH 7.6). The ImmPRESS polymer reagent kit (Vector Laboratories, Burlingame, CA) was used for detection of bound primary antibodies which were visualized using the SK-4100 DAB reagent (Vector Laboratories). All slides were digitally scanned using an Aperio ScanScope CS instrument (40× magnification; Aperio Technologies Inc., Vista, CA), and images of representative areas of pathology were captured using the ImageScope software (40× magnification; Aperio Technologies Inc.).

Quantitative analysis

For western blot data, soluble and insoluble bands were quantified using ImageJ software (NIH, Bethesda, MD) and the fractions of insoluble αsyn were calculated using the following equations: P/S + P or Triton insoluble/Triton soluble + Triton insoluble. Densitometric comparisons were performed in GraphPad Prism software using one-way analysis of variance (ANOVA), with post hoc analysis using Dunnett’s test to compare combination of aggregation conditions to the FL mouse αsyn, 21-140 αsyn mPFFs or m21-140 αsyn 1-135 mPFFs control depending on the experiments. For SDS-PAGE data, readings for each replicate sample averaged before using two-way analysis of variance (ANOVA), with post hoc analysis using Dunnett’s test to compare fraction of insoluble αsyn of each truncation at each time point to the FL control.

For comparison of inclusion pathology in mPFF injected mice, for each cohort (n = 8) the number of cellular inclusions (small neurites were not counted) for a single 5 μm section within the anterior hippocampus (coordinates from Bregma: anterior/posterior −2.2 mm) ipsilateral to the injection site were counted. The number of cellular inclusions within the ipsilateral cortex on the same section were similarly counted; inclusions were counted to a maximum value of 70. The number of inclusions within each region for each cohort was compared using one-way analysis of variance (ANOVA), with post hoc analysis using Dunnett’s test to compare the 1-125 and 1-129 mPFF injected mice with FL mPFF injected mice.

Results

C-truncation of mouse αsyn promotes formation of insoluble fibrils

Monomers of αsyn have previously been shown to aggregate into fibrils when agitated at physiologic temperatures (27). Six of the most common C-truncated forms of mouse αsyn (1-115, 1-119, 1-122, 1-124, 1-125 and 1-129) (5, 7, 28) were assessed for their polymerization into amyloid fibrils compared with FL mouse αsyn using biochemical insolubility analysis as previously described (7); this method was more suitable than fluorometry for the lower concentrations used to study the more aggregation prone forms of mouse αsyn relative to human αsyn (7). Monomers of each αsyn protein were diluted to 100 μM in PBS and incubated with agitation at 37°C; at time points of 0, 24, 48, and 96 hours samples were withdrawn from each tube (4 replicates per sample) and subjected to the insolubility analysis. At 0 hours, all αsyn proteins were fully soluble (Fig. 1). By 24 hours, the more extensively C-truncated αsyn species (1-115, 1-119, 1-122, 1-124, 1-125) were almost fully insoluble upon centrifugation compared with FL mouse αsyn only at ~35% insoluble (Fig. 1, Table 1). At 48 hours, all C-truncated species except the least truncated form (1-129) were almost fully sedimented into insoluble species compared with FL mouse αsyn which was ~40% insoluble (Fig. 1). By 96 hours, all mouse αsyn species were almost fully sedimented and insoluble, although the 1-119, 1-122, and 1-124 demonstrated a slight but significant increase in insoluble species compared to FL αsyn (Fig. 1, Table 1). Statistics are shown for all time points showing that most of these C-truncated forms of αsyn aggregate much more readily than mouse FL αsyn, in particular with greater C-truncation (Table 1).

Figure 1. C-truncations of mouse αsyn accelerate pathologic aggregation in vitro.

(A) Coomassie Blue R-250 stained SDS-PAGE gels showing select purified C-truncated forms of mouse αsyn (1-115, 1-122, 1-129) compared to FL mouse αsyn at time points of 0 and 48 hours (shaking at 37 °C) and fractionated into soluble (S) and pelleted (P) fractions. The apparent mobility of molecular mass markers (kDa) are displayed on the left. (B) Quantification of the fraction of insoluble αsyn for each of 6 truncated forms (1-115, 1-119, 1-122, 1-124, 1-125 and 1-129) of mouse αsyn compared to mouse FL αsyn at multiple time points (n = 4, error bars = s.d.). Two-way ANOVA and Dunnet’s multiple comparisons test was used to compare the insoluble fraction for tested truncated forms of αsyn with FL αsyn at each time-point; indicators of significance are shown, and no indication means no significant difference was observed. * = p ≤ 0.05, ** = p ≤ 0.01, **** = p ≤ 0.0001 (C) Western blot of C-truncated and FL forms of recombinant mouse αsyn (200 ng per lane) utilized in this study. Antibody SNL4 against residues 2-12 of αsyn was used.

Table 1. Statistical summary of in vitro aggregation rates of various forms of mouse αsyn utilizing biochemical fractionation analysis.

The fraction of insoluble FL αsyn following centrifugation (Fig. 1B) for each C-truncated mouse αsyn aggregation reaction at each time point was compared to that of FL mouse αsyn using two-way ANOVA and Dunnet’s multiple comparisons test.

αsyn truncations	Time (hours)
	0	24	48	96
	2-way ANOVA multiple comparisons test compared with FL αsyn
1-115	NS	****	****	NS
1-119	NS	****	****	*
1-122	NS	****	****	**
1-124	NS	****	****	*
1-125	NS	****	****	NS
1-129	NS	*	*	NS

NS = no significance,

^*= p ≤ 0.05,

^**= p ≤ 0.01,

^****= p ≤ 0.0001.

C-truncated forms of mouse αsyn readily aggregate in HEK293T cells upon prion-like seeding

The aggregation propensity of 8 of the most common C-truncated forms of αsyn (1-115, 1-119, 1-122, 1-124, 1-125, 1-129, 1-133 and 1-135) were assessed using a previously characterized HEK293T cell seeding assay (7, 29). These C-truncated forms of αsyn were previously studied in human αsyn, and here are generated in mouse αsyn which is also more prone to aggregation due to the presence of the A53T mutation (4, 27, 30). Upon transfection, HEK293T cells expressed each of the C-truncated forms of mouse αsyn when analyzed at 48 hours post transfection and all these proteins were soluble after Triton X-100 extraction similar to FL mouse αsyn if mPFFs were not added (Fig. 2A). For exogenous seeding studies, one hour after transfection mPFFs comprised of 21-140 mouse αsyn were added to cells expressing the C-truncated forms of mouse αsyn as these fibrils are not detected with the N-terminal specific antibody SNL4 (7); in the presence of these exogenous fibrils resulting in prion-like conformational templating and aggregation (22), it is demonstrated through western blotting that at 48 hours post transfection all of the C-truncated forms of αsyn aggregate to a significantly greater extent than FL mouse αsyn as evidenced by the increased fraction of insoluble αsyn (Fig. 2).

Figure 2. Aggregation of mouse αsyn is enhanced upon C-terminal truncation in cultured HEK293T cells with prion-like seeding.

(A) Western blots of FL and C-truncated forms of mouse αsyn expressed in HEK293T cells with no added fibrils; no insoluble αsyn forms without prion-like seeding. Antibody SNL4 against residues 2-12 of αsyn was used. The apparent mobility of molecular mass markers (kDa) are displayed on the left. (B) Western blots displaying biochemical Triton X-100 soluble and insoluble fractions of αsyn from each cell culture sample (n = 3) expressing the indicated truncated form of mouse αsyn followed by treatment with exogenous 1 μM 21-140 mPFFs. SNL4 against residues 2-12 of αsyn was used as it does not detect the 21-140 mPFFs. The apparent mobility of molecular mass markers (kDa) are displayed on the left. (C) Densitometric analysis of the blots in B (n = 3, error bars = s.d.); one-way ANOVA with Dunnet’s test was utilized to compare the final extent of aggregation for C-terminal truncated forms of mouse αsyn and FL mouse αsyn. NS = no significance, **** = p ≤ 0.0001.

Mouse PFFs comprised of C-truncated αsyn functionally alters prion-like seeding of FL αsyn in HEK293T cells and primary neuronal cultures

Recent structural studies suggest that C-terminal truncation could modulate the structure of αsyn fibrils such that a species like barrier could develop in which seeding of FL mouse αsyn by C-truncated forms of αsyn would be impaired (9–11). To assess this hypothesis using the previous HEK293T cell experimental paradigm, mPFFs comprised of C-terminally truncated αsyn were treated to cells expressing FL mouse αsyn. Antibody 94-3A10 that does not detect C-terminally truncated αsyn was used to monitor the formation of insoluble FL mouse αsyn (Fig. 3). Upon investigation, a trend was observed whereby lesser C-terminal truncation (1-133 and 1-135) does not significantly alter seeding of FL mouse αsyn, however further C-terminal truncation (1-124, 1-125, and 1-129) significantly decreases prion-like seeding of FL mouse αsyn, and extensive C-terminal truncation (1-115, 1-119, 1-122) leads to almost complete elimination of cross-seeding of FL mouse αsyn (Fig. 3). Fibrils generated to contain both 1-135 and one of 1-115, 1-119, or 1-122 in a 1:1 ratio were able to seed FL mouse αsyn, but not as efficiently as 1-135 mPFFs alone (Fig. 3). In order to confirm these finding in neurons, primary neuronal-glial cultures (PNCs) were generated from E16 mice; these PNCs were similarly incubated with the same C-truncated mPFFs and subjected to the same biochemical fractionation analysis at 10 days post fibril addition. The trend seen for seeding with C-truncated mPFFs was almost identical to that observed in the HEK293T cell assay; seeding is slightly impaired upon moderate truncation (1-124) and almost abolished in the most C-truncated forms (1-115, 1-119, 1-122) (Fig. 4).

Figure 3. mPFFs comprised entirely of C-truncated αsyn become less efficient at seeding FL mouse αsyn in cultured HEK293T cells.

(A) Western blots displaying biochemical Triton X-100 soluble and insoluble fractions for HEK293T cells transfected to express FL mouse αsyn (n = 3) and treated with 1 μM of indicated C-truncated mPFFs. Antibody 94-3A10 against residues 130-140 of αsyn was used for all western blots except for the 21-140 mPFFs added to FL αsyn which was analyzed with antibody SNL4 against residues 2-12. The apparent mobility of molecular mass markers (kDa) are displayed on the left. (B) Densitometric analysis of western blots in A where cells expressing mouse FL αsyn were treated with mPFFs comprised of individual mouse C-truncated asyn proteins as indicated (n = 3, error bars = s.d.); one-way ANOVA with Dunnet’s test was utilized to compare the seeding ability of increasingly C-truncated mPFFs with 21-140 mPFFs. (C) Densitometric analysis of western blots in A with seeding experiments utilizing mixed mPFFs containing 1-135 and other mouse αsyn C-truncations as indicted (n = 3, error bars = s.d.); one-way ANOVA with Dunnet’s test was used to determine the mixed mPFFs seeding ability compared with 1-135 mPFFs. * = p ≤ 0.05, *** = p ≤ 0.001, **** = p ≤ 0.0001.

Figure 4. mPFFs comprised entirely of C-truncated αsyn are less efficient than FL mouse αsyn at seeding endogenous FL mouse αsyn in cultured murine primary neuronal-glial cultures.

(A) Western blots depicting Triton X-100 soluble and insoluble biochemical fractions from E16 neuronal-glial cultures (n = 3) treated with 1.5 μM of C-truncated mPFFs. Antibody 94-3A10 against residues 130-140 of αsyn was used for all western blots. Fibrils comprised entirely of more extensively C-truncated mouse αsyn are less efficient at seeding FL mouse αsyn compared with control 1-135 mPFFs. The apparent mobility of molecular mass markers (kDa) are displayed on the left. (B) Densitometric analysis of the blots in A (n = 3, error bars = s.d.); one-way ANOVA with Dunnet’s test was used to compared the seeding ability of increasingly C-truncated mPFFs with 1-135 mPFFs. NS = no significance, * = p ≤ 0.05, **** = p ≤ 0.0001.

Comparison of mPFFs comprised of FL and C-truncated mouse αsyn in seeding pathology in mice upon intra-hippocampal injection

In order to assess the relative abilities of C-truncated mPFFs to induce pathology of endogenous mouse FL αsyn in vivo, mice were unilaterally injected into the anterior hippocampus with mPFFs comprised of either 1-125, 1-129, or FL mouse αsyn (n = 8 mice per group). A cohort of 3 mice was similarly injected with PBS. At 3 months post-injection, these mice were sacrificed and their brains subject to histologic analysis with antibodies selective for aggregated αsyn (Fig. 5, Table 2). Injected mPFFs comprised of recombinant proteins are not pSer129 positive (31), allowing detection of only endogenously formed αsyn aggregates using antibody 81A (Fig. 5). As expected from HEK293T and PNC seeding experiments, the 1-125 αsyn mPFFs were less efficient in seeding endogenous mouse FL αsyn as only 5/8 mice displayed inclusion pathology that was mostly limited to the injection site (anterior hippocampus, ipsilateral) with approximately 2-5 inclusions per 40x visual field (Fig. 5). Additional pathology was seen in 3/8 mice in the posterior hippocampus, and only rare inclusions were seen in 1/8 mice in the cortex adjacent to the injection site. For the lesser truncated 1-129 αsyn mPFFs, 7/8 mice displayed moderate pathology in both the anterior and posterior hippocampus ipsilaterally; in both cohorts 5-15 inclusions per 40x visual field were observed in these regions (Fig. 5). The biggest difference between the 1-125 and 1-129 αsyn mPFFs injected cohorts was observed in the ipsilateral cortex adjacent to the anterior hippocampus, where extensive inclusion development (5-15 inclusions per 40x visual field) was observed in 6/8 1-129 αsyn mPFFs injected mice whereas only 1 of the 1-125 αsyn mPFFs injected mice displayed rare cortical pathology (Fig. 5). The mice injected with FL αsyn mPFFs were overall similar to the 1-129 αsyn mPFFs cohort in regional distribution and density of pathology; 7/8 mice displayed pathology ipsilateral to the injection site in the anterior and posterior hippocampus and 7/8 for the cortex adjacent to the injection site (Fig. 5, Table 2). Quantitatively, 1-125 αsyn mPFF injected mice contained less pSer129 (81A) positive αsyn inclusions in both the anterior hippocampus and adjacent cortex ipsilateral to the injection site compared with 1-129 and FL αsyn mPFF injected mice (Fig. 5). No pathologic inclusion formation was observed in the 3 mice injected with PBS alone (Fig. 5, Table 2). These results further demonstrate that the C-terminus of αsyn is important in functionally determining prion-like properties of pathologic αsyn, and removal of more than ~10-15 residues from the C-terminus results in lessened seeding of the endogenous FL mouse αsyn.

Figure 5. mPFFs comprised of extensively C-truncated mouse αsyn seed pathology in mice albeit less efficiently than FL mPFFs.

αsyn inclusion pathology in mice following anterior intra-hippocampal injection of 4 μg of mPFFs comprised of 1-125, 1-129, or FL mouse αsyn. Regions from control mice injected with PBS are also shown. Images display αsyn inclusions in the ipsilateral anterior and posterior hippocampus along with cortex adjacent to the injection site as pathology is mainly limited to these regions in PFF injected mice. (A) Demonstration of inclusions using antibody 81A specific for pSer129 αsyn which is a marker of pathologic αsyn. (B) Detection of inclusions using antibody 94-3A10 raised against residues 130-140 of αsyn. With both antibodies, pathology is compared in density and distribution between the cohorts. Scale bar is 50 μm. (C) Quantitation and comparison of total number of 81A positive cellular inclusions within the anterior hippocampus for all mPFF injected mice (n = 8 each mPFF type, error bars = s.d.); one-way ANOVA with Dunnet’s test was used to compare the number of inclusions within 1-125 mPFF and 1-129 mPFF injected mice with FL mPFF injected mice. NS = no significance, * = p ≤ 0.05. (D) Quantitation and comparison of total number of 81A positive cellular inclusions within the cortex adjacent to the injection site for all mPFF injected mice (n = 8 each mPFF type, error bars = s.d.); one-way ANOVA with Dunnet’s test was used to compare the number of inclusions within 1-125 mPFF and 1-129 mPFF injected mice with FL mPFF injected mice. A maximum of 70 inclusions were counted for each mouse. NS = no significance, ** = p ≤ 0.01.

Table 2. Summary of pathologically afflicted regions in mice following intra-hippocampal injection with mPFFs.

For each cohort of 8 mice, the number of mice displaying αsyn inclusions in each region of the brain analyzed is shown. The 3 control mice injected with PBS demonstrate no αsyn inclusion pathology.

Injection	Regions with αsyn pathology (ipsilateral to injection site)
	Anterior Hippocampus	Posterior Hippocampus	Cortex
1-125 αsyn	5/8	3/8	1/8
1-129 αsyn	7/8	7/8	6/8
FL αsyn	7/8	7/8	7/8
PBS	0/3	0/3	0/3

Discussion

C-terminal truncation of αsyn has received considerable attention due to the increased pathologic aggregation that these forms of αsyn display relative to FL αsyn (7). Indeed, C-truncated forms of αsyn are present in human disease (5, 20, 28) and were found to be particularly common within gastrointestinal regions in PD (i.e. the vermiform appendix) where PD is postulated to begin, suggesting that these aggregation prone forms of αsyn may be important in initial disease pathogenesis (32). In vitro, it was further shown that the presence of C-truncated αsyn can stimulate the aggregation of FL αsyn (7), indicating that in early fibril forming events the presence of truncated forms of αsyn may be a critical step in pathogenesis. What has received less attention is how C-terminal truncation of PFFs modulates prion-like seeding activity; multiple recent studies have demonstrated that PFFs are initially trafficked to lysosomes where extensive truncation is occurring at the exposed amino and carboxy terminal regions (2, 14, 15). These studies show that αsyn fibrils are C-truncated upon entry to neurons and other cells, thus the predominant pathologic αsyn species in early neuronal seeding events are heavily C-truncated. Recent structural studies suggest that C-terminal truncation changes the structure of αsyn fibrils (9–11), and likewise experiments utilizing various models have demonstrated functional consequences for seeding with different C-truncated forms of human αsyn (7, 8, 15).

Mouse and human αsyn differ by 7 amino acids, particularly in the C-terminal region where 5 on these alterations are located (33). Most importantly, mouse αsyn has been shown to aggregate far more readily than human αsyn due primarily to the presence of an alanine to threonine difference at residue 53 which if present in humans leads to familial synucleinopathy (4, 34, 35). Mouse αsyn has been postulated to form structurally different fibrils compared with human αsyn (4, 33, 36, 37), and the mixture of both mouse and human αsyn within the same disease model may complicate interpretation of results as has been shown for prior seeding and viral transduction experiments where inhibitory effects to aggregation and seeding often result (4, 27, 38, 39). It is important to understand the aggregation and spread of pathologic αsyn with both mouse and human αsyn individually, as mouse αsyn is typically used for modeling synucleinopathy (PFF injection models into NTG mice) without αsyn overexpression and these mouse models can recapitulate many cardinal symptomatic and pathologic features of PD and LBD (2, 40, 41).

Herein, it was demonstrated that C-truncation of mouse αsyn increases aggregation propensity similar to observations for human αsyn. The more extensively C-truncated forms of murine αsyn are more likely to spontaneously self-aggregated and be seeded in the presence of PFFs. These findings are consistent with the notion that C-truncation of αsyn eliminates acidic residues present within the C-terminus of both mouse and human αsyn resulting in increased hydrophobicity and aggregation propensity (7, 8). Compared with human αsyn, even slight C-terminal truncation of mouse αsyn appears to greatly accelerate aggregation into fibrils; the combination of the residue 53 threonine amino acid difference in mouse versus human αsyn and the loss of protective negative charge in the C-terminus together renders these forms of mouse αsyn particularly prone to aggregation. Importantly, we demonstrate the functional consequences of C-truncation of αsyn on prion-like seeding activity in PNCs and in vivo in mice brains that are best studied with mouse αsyn due to the species barrier phenomenon in seeding (4). In all 3 assays utilized (HEK293T seeding, PNC seeding, and intra-hippocampal injection), progressive C-truncation leads to a significant decrease in cross-seeding of FL αsyn after 10-15 C-terminal residues are lost. A similar phenomenon was seen when using truncated human PFFs injected into mice (8). This loss of seeding activity is likely due to the aforementioned structural changes in the fibrils, demonstrating that even though the C-terminus is not found within the core amyloid structure of fibrils C-terminal alterations can still functionally impact the structure in conformational templating ability when interacting with endogenous αsyn monomers. However, prion-like seeding activity is only slightly lessened when the PFFs are comprised of only partially C-truncated αsyn, which may be the more physiologically relevant species (7). Thus, it is likely that the partial carboxyl processing of αsyn by lysosomal activities generates seeds with diverse activities akin to αsyn prion strains and this hypothesis will further investigated in future studies. Importantly, alteration in prion-like seeding may also occur upon insertion of various C-terminal tags to αsyn such as those used for fluorescent tracking, and caution should be exercised in the utilization of these modifications.

It is becoming apparent that the ramifications of C-terminal truncation are twofold: firstly, removal of negative charge and protective motifs upon C-truncation greatly accelerates fibril formation from monomeric C-truncated αsyn which may be important in initial fibril formation and has been well studied (7, 8, 42, 43). Secondly, C-terminal truncation changes the fibril structure such that cross-seeding of the FL αsyn monomers is slowed, which is difficult to interpret in disease context. If all αsyn fibrils are indeed trafficked to lysosomes and subsequently heavily C-truncated upon initial neuronal entry, this may partially explain why the time course of synucleinopathies occur over decades as opposed to the rapid appearance of pathology and symptoms seen in αsyn transgenic mice where overexpression of αsyn may dampen the consequences of incompatible fibril structure. In human disease, it could be that extensively C-truncated fibrils must seed endogenous FL αsyn monomers in a slow process before robust fibril formation occurs within the neuron leading to toxicity and extra-cellular release of fibrils where the cycle begins anew in the next neuron. Additionally, if αsyn fibrils are becoming only partially C-truncated (not every αsyn protein within fibrils are C-truncated) due to limited proteolytic activity, the seeding activity of αsyn fibrils may not be impaired or even increased which has been previously shown (7–9, 15, 44). It is possible that C-terminal truncation of αsyn is both harmful in initial fibril forming events, and also protective in dampening the speed with which prion-like spread occurs throughout the nervous system. Overall, this study demonstrates that αsyn C-terminal modifications functionally alter the prion-like seeding activity of PFFs in some of the most common models of synucleinopathy used, which will be of value in understanding how synucleinopathies progress.

Abbreviations:

The abbreviations used are:

Αsyn

α-synuclein

ANOVA

analysis of variance

C

carboxy

DAB

3, 3- diaminobenzidine

DMEM

Dulbecco’s Modified Eagle’s Medium

FBS

fetal bovine serum

FL

full-length

LB

Lewy body

LBD

Lewy body dementia

NTG

non-transgenic

PBS

phosphate buffered saline

pSer129

phosphorylated Ser129 α-synuclein

PD

Parkinson’s disease

P

pelleted

PFFs

pre-formed fibrils

PNC

primary neuronal-glial culture

S

soluble

TBS

Tris buffered saline