Induction of De Novo Α-Synuclein Fibrillization in a Neuronal Model for Parkinson's Disease

Induction of de novo α-synuclein fibrillization in a neuronal model for Parkinson’s disease

Mohamed-Bilal Faresa, Bohumil Macoa, Abid Oueslatia,1, Edward Rockensteinb, Natalia Ninkinac, Vladimir L. Buchmanc, Eliezer Masliahb, and Hilal A. Lashuela,d,2

aBrain Mind Institute, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, CH-1015, Switzerland; bDepartment of Neurosciences, University of California San Diego, La Jolla, CA 92093; cSchool of Biosciences, Cardiff University, Cardiff, CF10 3AX, United Kingdom; and dQatar Biomedical Research Institute, Hamad Bin Khalifa University, Doha, 5825, Qatar

Edited by Thomas C. Südhof, Stanford University School of Medicine, Stanford, CA, and approved January 4, 2016 (received for review July 25, 2015) Lewy bodies (LBs) are intraneuronal inclusions consisting primarily periments in mice supported this suggestion, as hα-Syn transgenic of fibrillized human α-synuclein (hα-Syn) protein, which represent (Tg) mice lacking endogenous mouse α-Syn (mα-Syn) expression the major pathological hallmark of Parkinson’s disease (PD). Al- exhibited exacerbated pathology compared with WT counterparts though doubling hα-Syn expression provokes LB pathology in humans, (5), and manifested fibrillar/granular accumulations in the olfac- hα-Syn overexpression does not trigger the formation of fibrillar tory bulb (6). Interestingly, in vitro test-tube experiments also LB-like inclusions in mice. We hypothesized that interactions between showed that small amounts of mα-Syn directly inhibit the fibrilli- exogenous hα-Syn and endogenous mouse synuclein homologs could zation of purified hα-Syn protein in solution (7). be attenuating hα-Syn fibrillization in mice, and therefore, we system- Despite these interesting observations, it remained unclear atically assessed hα-Syn aggregation propensity in neurons derived whether endogenously expressed synuclein homologs directly from α-Syn–KO, β-Syn–KO, γ-Syn–KO, and triple-KO mice lacking ex- inhibit hα-Syn aggregation in neurons, and whether ectopic pression of all three synuclein homologs. Herein, we show that hα-Syn hα-Syn expression in their absence would allow de novo hα-Syn forms hyperphosphorylated (at S129) and ubiquitin-positive LB-like in- fibrillization events. Therefore, we systematically evaluated the clusions in primary neurons of α-Syn–KO, β-Syn–KO, and triple-KO propensity of hα-Syn to aggregate on synuclein-KO backgrounds. mice,aswellasintransgenicα-Syn–KO mouse brains in vivo. Impor- Using a battery of biochemical and imaging techniques, we dem- tantly, correlative light and electron microscopy, immunogold labeling, onstrate that in cultured primary neurons and brains of mα-Syn– and thioflavin-S binding established their fibrillar ultrastructure, and KO mice, overexpressed hα-Syn aggregates readily into inclusions fluorescence recovery after photobleaching/photoconversion experi- that are similar to LBs in terms of solubility, immunoreactivity, and ments showed that these inclusions grow in size and incorporate amyloidogenicity, and represent a bona fide fibrillization process as soluble proteins. We further investigated whether the presence of revealed by serial-section transmission electron microscopy (ssTEM), α homologous -Syn species would interfere with the seeding and live imaging, and response to pharmacological aggregation inhibitors. α − − spreading of -Syn pathology. Our results are in line with increasing Similarly, primary neurons lacking expression of β-Syn (β-Syn / )or α evidence demonstrating that the spreading of -Syn pathology is most of all three homologs (α-, β-, and γ-Syn) also manifest enhanced hα- prominent when the injected preformed fibrils and host-expressed α Syn aggregation, thereby suggesting that endogenous synuclein -Syn monomers are from the same species. These findings provide homologs may represent natural regulators of abnormal α-Syn ag- insights that will help advance the development of neuronal and α gregation. To dissect potential mechanisms underlying this phe- in vivo models for understanding mechanisms underlying h -Syn nomenon, we performed immunoprecipitation and surface plasmon intraneuronal fibrillization and its contribution to PD pathogenesis, and for screening pharmacologic and genetic modulators of α-Syn fibrillization in neurons. Significance

Parkinson’s disease | alpha-synuclein | aggregation Although it has been established for over 100 years that Lewy bodies (LBs) represent the major pathological hallmark of Parkin- son’s disease (PD), we still do not know why these fibrillar intra- he aggregation of proteins into fibrillar structures is a key neuronal inclusions of α-synuclein (α-Syn) protein form, or how hallmark of many neurodegenerative disorders. In Parkin- T they contribute to disease progression. One of the major causes son’s disease (PD), α-synuclein (α-Syn), a predominantly pre- underlying this gap in knowledge is the lack of animal models that synaptic protein involved in the regulation of neurotransmitter reproduce the formation of fibrillar LB-like inclusions. In this study, release, abnormally fibrilizes and forms intraneuronal inclusions we show that the absence of human α-Syn (hα-Syn) fibrillization termed “Lewy bodies” (LBs) (1, 2). So far, the mechanisms un- into LBs in mice can be attributed to interactions between hα-Syn derlying LB formation remain poorly understood, and the impact and its endogenously expressed mouse α-Syn homologue. More- of LB presence on neuronal viability remains controversial, in over, we provide well-characterizedprimaryneuronalandinvivo part due to the lack of animal models recapitulating α-Syn models that recapitulate the main molecular feature of PD, bona fibrillization into LB-like inclusions in the brain. fide α-Syn fibrillization. Because patients with familial history of parkinsonism were

found carrying either multiplications or point mutations of the Author contributions: M.-B.F., B.M., A.O., E.R., E.M., and H.A.L. designed research; M.-B.F., α-Syn gene SNCA (2), most animal models of PD have been B.M.,A.O.,andE.R.performedresearch;E.R.,N.N.,V.L.B.,andE.M.contributednew generated by overexpressing WT human α-Syn (hα-Syn) or mu- reagents/analytic tools; M.-B.F., B.M., A.O., E.R., N.N., V.L.B., E.M., and H.A.L. analyzed tant forms linked to familial PD (3). Strikingly, although rodent data; and M.-B.F., N.N., V.L.B., and H.A.L. wrote the paper. models expressing hα-Syn do not recapitulate the formation of The authors declare no conflict of interest. fibrillar LBs within dopaminergic neurons, hα-Syn overexpression This article is a PNAS Direct Submission. Drosophila 1Present address: Centre de Recherche du Centre Hospitalier de Québec, Axe Neurosci- in led to dramatic neuronal loss accompanied with ’ α Drosophila ence et Département de Médecine Moléculaire de l Université Laval, Québec, Canada LB-like structures comprising fibrillar -Syn (4). , un- G1V 4G2. α like rodents, do not express an endogenous -Syn homolog, 2To whom correspondence should be addressed. Email: [email protected]. α implying that h -Syn fibril formation could be more favorable This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. in models lacking endogenous α-Syn expression. Subsequent ex- 1073/pnas.1512876113/-/DCSupplemental.

E912–E921 | PNAS | Published online February 2, 2016 www.pnas.org/cgi/doi/10.1073/pnas.1512876113 Downloaded by guest on September 30, 2021 resonance (SPR) experiments, which showed that mα-Syn interacts nuclear and cytoplasmic membranes (Fig. 1A and Movie S1). A PNAS PLUS preferentially with aggregated hα-Syn preformed fibrils (PFFs) similar phenotype was observed in primary neurons derived from − − tm1Rosl rather than with monomers. Importantly, in vitro aggregation another SNCA / mouse strain (B6,129 × 1-Snca /J) generated experiments and in vivo assessment of cross-seeding pro- by targeted deletion of two exons of the mα-Syn gene (Fig. S1 C pensities of hα-Syn and mα-Syn showed that the observed cross- and D) (13), thereby establishing that the formation of inclusions species interactions attenuate seeding and spreading of aggre- is directly linked to the specific loss of mα-Syn expression. In line − − gates. These findings possibly explain why current rodent PD with this finding, re-expression of mα-Syn in SNCA / neurons α α models expressing h -Syn do not exhibit pronounced h -Syn significantly reduced the formation of intraneuronal hα-Syn in- fibrillization, and provide models that reproduce a critical clusions, and restored the diffuse distribution of hα-Syn in most pathological feature of the disease: the de novo formation of B α neurons (Fig. 1 ). fibrillar h -Syn aggregates. To investigate whether the total abolishment of mα-Syn ex- α Results pression is necessary to promote h -Syn inclusion formation, we − − assessed whether decreasing mα-Syn levels in WT neurons via Induction of hα-Syn Inclusion Formation in SNCA / Primary Neurons. Several studies have reported that overexpressed hα-Syn in shRNA-mediated silencing is sufficient to promote this process. mouse primary neurons exhibits diffuse localization without forming Transient expression of three different vectors encoding shRNA α discreet inclusions (8–11). To assess whether the absence of mα-Syn hairpin loops showed efficient silencing of m -Syn in transfected would affect this distribution, we examined the localization of neurons as compared to neurons transfected with scrambled transiently expressed hα-Syn in primary neurons derived from shRNA sequences (Fig. S1E), and biochemical analysis of lenti- − − C57Bl6/J/ola/hsd mice (SNCA / ) lacking expression of mα-Syn virally infected neurons showed ∼50% reduction in mα-Syn ex- (Fig. S1 A and B) (12). Interestingly, although hα-Syn exhibited the pression (Fig. S1F). Notably however, mα-Syn silencing did not previously reported diffuse distribution in WT neurons, a significant promote the formation of hα-Syn inclusions in WT neurons (Fig. − − proportion of the SNCA / neurons developed spheroid-like in- S1G), suggesting that even low levels of mα-Syn are sufficient to clusions that were consistently observed in close association with attenuate inclusion formation. NEUROSCIENCE

Fig. 1. The absence of mα-Syn promotes hα-Syn aggregation in primary neurons. (A)hα-Syn exhibits diffuse distribution in WT neurons and forms inclusions − − (>1 μm) in cell bodies and neurites in a significant proportion of SNCA / neurons. β3-Tubulin staining was performed to reveal neurons, and merged images − − show orthogonal Z-stack projections (Orth. Proj.). (B) Significantly fewer inclusions (>1 μm) are observed in SNCA / neurons with restored mα-Syn expression. − − (C) Significantly less monomeric hα-Syn is detected in nonionic detergent-soluble (Det. Sol.) fractions of lentivirally infected SNCA / neurons than in WT − − counterparts (n = 4 independent experiments). In contrast, nonionic detergent-insoluble (Det. Insol.) fractions of infected SNCA / neurons show more monomeric and HMW hα-Syn species, comparable to the detergent-insoluble fractions of WT neurons treated with hα-Syn PFFs. Actin was used to control for equal protein loading. In B and C,hα-Syn and mα-Syn were differentially revealed using the Syn-211 and D37A6 antibodies, respectively. In A–C, the Mann–Whitney test was applied to obtain P values. In A and B, 25 neurons per condition were quantified (n = 3 independent experiments). (D) Nine different anti–α-Syn antibodies (epitopes in − − − − Table S1) detect inclusions in SNCA / neurons transfected with hα-Syn. (E)hα-Syn inclusions in transfected SNCA / neurons are ThS-positive, phosphorylated at S129, and ubiquitinated. Colocalization was confirmed by assessing regression coefficients (R2). (Scale bars: 10 μminA and B and 5 μminD and E.)

Fares et al. PNAS | Published online February 2, 2016 | E913 Downloaded by guest on September 30, 2021 − − hα-Syn Inclusions Reproduce Key LB Features and Exhibit Fibrillar One possibility is that the inclusions in SNCA / neurons Ultrastructure. Several cellular models manifesting α-Syn accumu- represent aggregated hα-Syn that is contained within vesicles lation into inclusions in human cell lines have been reported, upon of the endo-lysosomal pathway. Therefore, we systematically α-Syn overexpression alone (14–16), coexpression with synphilin-1 assessed the colocalization of inclusions with specific markers for (17), exposure to proteasome inhibitors (9), or application of early endosomes [Ras-related protein (Rab) 5 and Ras homolog oxidative/nitrative insults (18, 19). Nevertheless, inclusions observed gene family member B (RhoB)], late endosomes (Rab7), lyso- under these conditions typically do not reproduce all key LB somes [lysosomal-associated membrane protein 1 (Lamp1)], features, including decreased solubility, hyperphosphorylation at autophagosomes [microtubule-associated proteins 1A/1B light Serine-129 (pS129) (20), ubiquitination (21), thioflavin-S (ThS) chain 3 (LC3)], and multilamellar bodies [CD63 antigen binding, and fibrillar ultrastructural organization (1). Therefore, (CD63)]. As shown in Fig. S4A, although some hα-Syn inclu- − − we assessed whether the hα-Syn inclusions observed in SNCA / sions showed weak partial colocalization with cotransfected neurons fulfill these pathological features. markers of late endosomes (Rab7-GFP) and lysosomes To determine whether the formation of inclusions is linked to (Lamp1-RFP) indicating potential degradation by this path- − − decreased hα-Syn solubility, WT or SNCA / neurons were infected way, most of the inclusions showed no colocalization with any with lentiviruses encoding hα-Syn and were then fractionated into of the cotransfected markers. − − nonionic detergent-soluble and nonionic detergent-insoluble Next, we assessed whether the inclusions formed in SNCA / − − fractions. Using this assay, lentivirally infected SNCA / neurons neurons are readily detectable using common LB probes, namely, showed significantly less monomeric hα-Syn in detergent-soluble antibodies against α-Syn (1), pS129–α-Syn (20), and ubiquitin fractions compared to WT counterparts (Fig. 1C). Interestingly, (21), as well as thioflavin-S (23). As shown in Fig. 1D, immu- this effect was not caused by decreased total hα-Syn expression in nostaining using nine different anti–α-Syn antibodies with epi- − − SNCA / neurons, because similar total hα-Syn RNA and pro- topes spanning most of the α-Syn sequence all revealed bright − − tein levels were observed in unfractionated WT and SNCA / spheroid intraneuronal inclusions, thereby establishing that these neurons (Fig. S2 A and B). In contrast, the decrease in soluble structures indeed comprise full-length hα-Syn. Moreover, dual monomeric hα-Syn was concomitant with the appearance of immunofluorescence analysis showed that the inclusions are high-molecular-weight (HMW) hα-Syn species in detergent- also hyperphosphorylated at S129 and are ubiquitinated, and − − insoluble fractions of lentivirally infected SNCA / neurons (Fig. costaining with thioflavin-S indicated that the inclusions could 1C). To validate our fractionation protocol, we treated WT comprise cross–β-sheet fibrillar content (Fig. 1E). neurons with insoluble hα-SynAlexa633 PFFs (characterized in Fig. To establish whether the hα-Syn inclusions comprise the S3), which showed a strong signal almost exclusively within the characteristic fibrillar ultrastructure observed in LBs, we per- detergent-insoluble fractions (Fig. 1C), as previously reported formed correlative light and electron microscopy (CLEM) which (22). Taken together, these results suggest that abolishing mα-Syn allows ultrastructural examination of neurons previously identi- expression enhances the aggregation propensity of hα-Syn in pri- fied by confocal microscopy as containing fluorescent inclusions mary neurons, as reflected by decreased solubility and the forma- (Fig. 2 A–C). Seventy-two hours posttransfection, hα-Syn in- tion of insoluble HMW hα-Syn aggregates. clusions were detected in close association with nuclear and

− − Fig. 2. Ultrastructure of hα-Syn inclusions in SNCA / neurons. (A) For CLEM analysis, a stained neuron was imaged by confocal microscopy and then was reprocessed and imaged by TEM, resulting in a high-resolution 3D acquisition of the neuron. For illustration, the nucleus is rendered in blue, the cytosol in purple, and inclusions in yellow. (Scale bars: 5 μm.) (B) To correlate structures observed by ssTEM and fluorescence, single-plane images were superimposed. High-resolution images of inclusions (red boxes 1 and 2) are shown in the lower panel, with arrowheads indicating filamentous structures. (Scale bars: 5 μmin the upper panel and 2 μm in the lower panel.) (C) Same analysis as in B on different confocal and ssTEM planes from the same neuron. (D) Most filaments observed by ssTEM (n = 473) have a width ranging from 7–13 nm. The frequency histogram and normal curve (in red) are shown, as well as values of mean ± − − SD. (E) Immunogold labeling of SNCA / neurons expressing hα-Syn with the ab-6176 anti-synuclein antibody establishes that filaments are positive for synuclein. Different magnifications of the inclusion (blue arrowhead) and filaments (green arrowheads) are shown. (Scale bar: 1 μm in the left panel and 125 nm in the middle and right panels.) (F) A 3D model of the inclusion (blue arrowhead in E) reveals a mesh of intertwining filamentous structures (in purple) with gold particles reacting with the inner and outer portions. The nucleus is rendered in blue, the inclusion in yellow, and immunogold particles in green. Different views of the inclusion are shown.

E914 | www.pnas.org/cgi/doi/10.1073/pnas.1512876113 Fares et al. Downloaded by guest on September 30, 2021 cytoplasmic membranes or near mitochondria but were rarely found mEOS2–α-Syn inclusions were detected 24 h posttransfection PNAS PLUS − − surrounded by a uniform lipid bilayer, thereby further ruling out in SNCA / neurons and continued to grow in volume over the possibility that the inclusions are vesicular in nature. Re- 72 h. Moreover, the detected inclusions were reactive with antibodies markably, high-magnification ssTEM of the correlated inclusions against total α-Syn, ubiquitin, and pS129–α-Syn (Fig. 3C), suggesting revealed intertwining whirls of filamentous structures in most that the fusion protein reproduces the aggregation properties of analyzed inclusions. Individual measurements of these structures untagged hα-Synandcouldbeusedtoinvestigatethekineticsof showed that the filaments have an average diameter ranging inclusion formation in neurons. from 7–13 nm (Fig. 2D), which is similar to that of fibrils within Short-interval (∼1 h) confocal live imaging followed by 3D genuine LBs (24), and thereby suggests that hα-Syn could be surface rendering showed that the volume of individual inclu- aggregating into fibrils within these inclusions. sions increased over time, with very few reversible fusion events To rule out the possibility that these flexible filamentous structures noted (Fig. 3D). Moreover, analysis of the speed of moving could represent membranes that have been entrapped within inclu- particles showed that most detected inclusions are immobile sions, we performed an additional CLEM experiment in which we (Fig. 3D), thereby ruling out the possibility of inclusion growth treated neurons exhibiting de novo aggregates (composed of being caused mainly by the fusion of small, motile aggregates. Myc–hα-Syn) with hα-SynAlexa633 PFFs for 24 h and then, Notably, the percent increase in volume was variable among within adjacent neurons, compared the ultrastructure and diameter different inclusions, with some doubling their volume within of de novo filaments (comprising only Myc–hα-Syn), internalized 50 min and others showing modest changes. This observation PFFs, mixtures of both, and intracellular membranes (Fig. S4 B and suggests that the growth rate of inclusions is not linear and constant C). As shown in Fig. S4 D and E, although intracellular membranes over time, and is not necessarily similar for the whole population of showed a broad distribution of diameters (7–12 nm with a mean inclusions within the same neuron. average of 8.7 nm), internalized PFFs and mixtures of de novo To determine whether mEOS2–α-Syn inclusions incorporate formed filaments and PFFs showed a much more defined width soluble hα-Syn over time, we performed fluorescence recovery distribution of 10–13 nm and a higher mean diameter of ∼11 nm. after photobleaching (FRAP) and photoconversion live-imaging Interestingly, although filaments in de novo inclusions exhibited experiments (Fig. 3E), which allow assessing the rates of protein a broader width distribution compared to PFFs and mixtures diffusion into (DIN) and out of (DOUT) inclusions respectively, (7–13 nm), with some of the filaments having a low diameter that and determining the relative proportion of immobile protein could classify them as membranous, the great majority (∼75%) within inclusions (immobile fraction, IF). In FRAP experiments

were in the range of 10–13 nm, within the narrow distribution of we photobleached individual inclusions and monitored the re- NEUROSCIENCE PFFs. covered fluorescence from diffusing soluble cytosolic protein. As To determine whether hα-Syn is the constituent of the ob- shown in Fig. 3F and Movie S2, FRAP measurements showed served fibrillar structures, we sought to assess their reactivity the recovery of fluorescent signal within photobleached inclu- with antibodies against α-Syn by immunogold labeling. First, we sions, and monoexponential fitting of FRAP plots from ∼90 probed the ability of four different antibodies that have different different inclusions allowed the estimation of DIN and IF. The 2 epitopes against the N terminus, C terminus, and non-Abeta average value for DIN was very low (∼0.03 μm /s), consistent with component (NAC) region of α-Syn to detect hα-Syn PFFs. No- previous results for immobilized α-Syn–tetracysteine inclusions tably, we found that polyclonal anti–PAN-Syn antibodies such as in SH-SY5Y cells (0.03–0.04 μm2/s) (26), suggesting the presence ab-6176 and FL-140 detect PFFs much more readily than of a structure that impedes free diffusion of soluble hα-Syn. In monoclonal hα-Syn–specific antibodies (Fig. S5A). Therefore, we line with this notion, ∼32% of the protein was estimated to be assessed the reactivity of the fibrillar structures observed within found within the IF, thereby establishing that inclusions com- −/− hα-Syn–expressing SNCA neurons to the ab-6176 antibody. prise immobilized proteins that are unable to equilibrate with the Importantly, 3D reconstruction of ssTEM images clearly showed cytoplasmic pool, likely because they are bound or sequestered in immunogold particles reacting with outer and inner portions a dense compact structure. The presence of high amounts of of the filaments detected within inclusions (Fig. 2 E and F), thereby soluble mobile protein (∼68%) within inclusions is in line with confirming the presence of synuclein within these structures. our ssTEM data showing filamentous structures embedded Taken together, these findings demonstrate that the inclusions within a bulk of electron-translucent milieu, which is probably −/− observed in SNCA neurons exhibit similarities to LBs in terms of sequestered soluble hα-Syn protein. solubility and immunoreactivity, and demonstrate signs of bona fide To assess the DOUT, we used the photoconvertible property of fibrillization events. However, the filaments do not have an orderly mEOS2, where we photoconverted individual inclusions by near- arrangement with a dense core, as typically observed in brainstem UV irradiation and monitored the decrease in photoconverted LBs, but are embedded among an electron-translucent medium, fluorescence intensity within inclusions. The decay in fluores- which is probably composed of sequestered soluble α-Syn protein. cence would reflect outward diffusion of proteins from the in- Because the filaments are not organized as in authentic LBs, they clusions to the cytosol. As expected, irradiation of inclusions may recapitulate early stages of LB formation that may need with a 405-nm laser resulted in rapid photoconversion of 488-nm more time or additional factors to mature and remodel into more mEOS2–α-Syn fluorescence to 568 nm (Fig. 3G and Movie S3). organized LB-like structures. Strikingly, although exponential fitting of decay curves from ∼50 different neurons revealed a slightly lower estimation of the IF − − hα-Syn Inclusions in SNCA / Neurons Grow in Size and Incorporate than obtained from FRAP experiments (16 ±10%), the esti- α α 2 Soluble -Syn. We then investigated mechanisms underlying h - mated DOUT (0.0022 ± 0.0007 μm /s) was almost 16-fold lower Syn inclusion growth over time using fluorescence microscopy. In H − − than the DIN (Fig. 3 ). This result suggests that, within the same SNCA / neurons transiently expressing hα-Syn, inclusions were time period, significantly more protein enters into inclusions detected 24, 48, and 72 h posttransfection, and the size of in- than diffuses out. As such, our findings together suggest that − − clusions appeared to increase over time as assessed by quantifying the hα-Syn aggregates in SNCA / neurons incorporate soluble 3D-rendered inclusion volumes (Fig. 3A). To investigate the ki- protein into stable structures, most likely the fibrils observed netics of inclusion growth in real time, we performed fluorescence by ssTEM. live-imaging experiments using hα-Syn that is N-terminally tagged to mEOS2, a fluorescent protein that is photoconvertible from bright Inclusion Formation in SNCA−/− Neurons Is an Aggregation-Driven green (506 nm) to orange-red (584 nm) when excited at near-UV Process. To establish whether the formation of hα-Syn inclusions wavelengths (25). As shown in Fig. 3B, somatic and neuritic is an aggregation-driven process, we assessed whether altering

Fares et al. PNAS | Published online February 2, 2016 | E915 Downloaded by guest on September 30, 2021 − − Fig. 3. hα-Syn inclusions in SNCA / neurons grow and incorporate soluble hα-Syn. (A) Immunofluorescence and 3D rendering of inclusions reveals a + significant increase in inclusion volume at 48 and 72 h posttransfection in 25 MAP2 neurons per condition. (B) Similarly, mEOS2–α-Syn inclusions (n = 50 per condition) show a significant increase in inclusion volume 72 h posttransfection. (C)mEOS2–α-Syn inclusions are immunopositive for total α-Syn (Syn-1), pS129–α-Syn (WAKO), and ubiquitin. (D)mEOS2–Syn inclusions are immobile and grow in volume during ∼1 h of live imaging. (Scale bars: 5 μminA–C and 2 μmin

D.) (E) FRAP and photoconversion experiments allow monitoring rates of Din and Dout of inclusions, respectively. (F and G, Upper) Fluorescence before, during, and after inclusion photobleaching (F) or photoconversion (G) is shown. Higher magnification images of yellow boxes are shown, with crossed circles denoting areas of photobleaching (F) or photoconversion (G). (Lower)Averageplots(± SD) of normalized FRAP or photoconversion recovery curves (n = 87 or 63 inclusions, respectively). (Scale bars: 2 μm at lower magnification and 1 μmathighermagnification.)Ch,channel.(H) Values for immobile fractions and diffusion coefficients obtained from photoconversion curves (n = 50 inclusions) are significantly lower than those obtained by FRAP. In A, B,andH,theMann–Whitney test was applied to obtain P values.

− − the propensity of hα-Syn for aggregation affects the formation of hα-Syn inclusions in SNCA / neurons is an aggregation- and/or growth of neuronal inclusions. Two modified hα-Syn driven process, as inclusion formation was significantly inhibited proteins, a C-terminally truncated variant (at position 120) that both by the introduction of a single amino acid substitution known has been shown to aggregate more readily than full-length to inhibit hα-Syn’s aggregation propensity, and by treatment with a α-Syn in vitro and in vivo (27), and a variant with the S87E known aggregation inhibitor. substitution that inhibits hα-Syn aggregation in similar models − − (28, 29), were expressed in WT or SNCA / neurons. As shown Endogenous β-Syn KO Is a Natural hα-Syn Aggregation Inhibitor in − − in Fig. 4A, although similar proportions of SNCA / neurons Neurons. Our results suggest that homologous members of the Δ expressing full-length (α-SynFL) or truncated (α-Syn 120)hα-Syn synuclein family could act as natural physiological inhibitors of developed inclusions, the S87E aggregation-deficient variant abnormal α-Syn aggregation. To test this hypothesis, we com- exhibited mostly diffuse distribution. This effect was not caused by a pared hα-Syn’s propensity for aggregation in primary neurons difference in protein expression levels among the three proteins, derived from mice lacking expression of β-Syn (β-Syn KO) (31), because similar mean fluorescence levels per neuron were detected γ-Syn (γ-Syn KO) (32), or all three synucleins (triple KO) (33). Δ across conditions (Fig. S5B). As expression of the α-Syn 120 mutant Loss of α- and β-Syn in corresponding KO cultures was validated did not affect the number of neurons forming inclusions, we in- by immunocytochemistry (Fig. 5A); γ-Syn expression was not vestigated whether the kinetics of mEOS2–α-Syn inclusion growth detectable in any of the cultures, probably because of its low are affected by this modification (Fig. 4B). Surface reconstruction of expression levels in hippocampal neurons, as previously reported Δ inclusions showed that at 24 h posttransfection mEOS2–α-Syn 120 (34). Remarkably, when we assessed the distribution of exoge- formed significantly larger inclusions than mEOS2–α-SynWT.Incon- nously expressed hα-Syn across the different cultures (Fig. 5B), trast, the few inclusions formed by the mEOS2–α-SynS87E mutant we found that β-Syn–KO and triple-KO neurons similarly de- were much smaller compared to mEOS2–α-SynWT inclusions at 48 h. velop α-Syn inclusions, unlike WT controls and γ-Syn–KO neu- Furthermore, we investigated whether treatment with a phar- rons that mostly exhibit diffuse hα-Syn distribution. Importantly, macological α-Syn aggregation inhibitor, tolcapone (30), would the inclusions formed in β-Syn–KO and triple-KO neurons were affect the formation of inclusions in this model. Remarkably, also hyperphosphorylated at S129, thus exhibiting a key patho- tolcapone treatment reduced the formation of inclusions by ∼60% logical LB-like phenotype (Fig. 5C). These findings suggest that, − − and restored the diffuse distribution of hα-Syn in SNCA / neurons unlike endogenous γ-Syn, the other two homologs, α-Syn (Fig. 4C). Together, these results demonstrate that the formation and β-Syn, have similar inhibitory effects on ectopic hα-Syn

E916 | www.pnas.org/cgi/doi/10.1073/pnas.1512876113 Fares et al. Downloaded by guest on September 30, 2021 were detected using three different antibodies against hα-Syn, PNAS PLUS thereby establishing their specificity (Fig. 6A and Fig. S6B). Moreover, the decrease in soluble hα-Syn was not the result of +/+ −/− decreased total hα-Syn expression in hα-Syn SNCA mice +/+ compared to hα-Syn counterparts, as similar total hα-Syn mRNA and protein levels were observed in unfractionated brains of both genotypes (Fig. S6 C and D). These findings suggestthathα-Syn aggregates readily and becomes less soluble in the absence of mα-Syn in Tg mouse brains. To evaluate further the extent of hα-Syn aggregation in mouse brains, we performed immunohistochemical analyses on sections treated with proteinase K, which allows selective visualization of highly structured proteinase K resistant aggregates. Consistent +/+ with previous studies (35), hα-Syn mice showed abundant intraneuronal α-Syn immunoreactive inclusions in the cortex, hippocampus, and striatum, some of which were proteinase K +/+ −/− resistant (Fig. 6B). Strikingly, neurons from hα-Syn SNCA mice showed higher immunoreactivity to anti–α-Syn antibodies and exhibited a significant increase in proteinase K-resistant inclusions in the cortex and hippocampus (Fig. 6B). Moreover, double immunolabeling showed that these inclusions are phosphorylated at S129 and exhibit strong reactivity for the synaptic vesicle protein synaptophysin (Fig. 6 C and D), both of which are markers of LB pathology (20, 36). Taken together, these findings demonstrate that the aggregation propensity of hα-Syn is enhanced in the absence of mα-Syn in vivo, as reflected by decreased hα-Syn solubility and accumulation into proteinase

K-resistant inclusions. NEUROSCIENCE To assess whether abolishing mα-Syn expression would promote the aggregation of human β-Syn (hβ-Syn), the considerably less aggregation-prone homolog of hα-Syn, we crossed Tg mice −/− +/+ expressing hβ-Syn (37) with SNCA mice to generate hβ-Syn −/− SNCA mice. Biochemical fractionation revealed that the absence of mα-Syn did not affect the levels of soluble hβ-Syn or β E − − cause the appearance of insoluble h -Syn species (Fig. S6 ). In Fig. 4. hα-Syn inclusion formation in SNCA / neurons reflects an aggregation- − − driven process. (A) Significantly fewer SNCA / neurons expressing α-SynS87E develop inclusions (>1 μm) than their α-SynFL and SynΔ120 counterparts (mean ± SD, n = 3 independent experiments with 25 neurons per condition). (B) Live imaging and 3D rendering of inclusions (50 neurons Δ per condition) show that mEOS2–α-Syn 120 forms significantly larger inclusions 24 h posttransfection than mEOS2–α-SynWT, and that the mEOS2–α-SynS87E − − mutant forms smaller inclusions at 48 h in SNCA / neurons. (C) Significantly − − fewer tolcapone-treated SNCA / neurons expressing hα-Syn (25 neurons per condition) develop inclusions (>1 μm) than DMSO-treated controls (mean ± SD, n = 3 independent experiments). In A the one-way ANOVA and Scheffé post hoc analysis were applied, and in B and C the Mann–Whitney test was applied to obtain P values. Orth. Proj., orthogonal projection. (Scale bars: 5 μm.)

aggregation. Our observation that similar percentages of β-Syn–KO and triple-KO neurons develop inclusions (∼35%) rules out the presence of a synergistic effect for the deletion of both proteins.

The Absence of mα-Syn Promotes Specific Aggregation of hα-Syn in Vivo. To test whether the presence of mα-Syn affects the aggregation propensity of hα-Syn in the brain, we crossed mice lacking endoge- −/− nous mα-Syn (SNCA ) (13) with mice carrying two copies of hα- +/+ +/+ −/− Syn (hα-Syn )(Fig. S6A)(35)togenerate(hα-Syn SNCA ) mice. Biochemical fractionation of brain homogenates into Fig. 5. Endogenous β-Syn naturally acts as an inhibitor of hα-Syn aggre- cytosolic (soluble) and particulate (insoluble) protein fractions was gation in primary neurons. (A) Immunocytochemistry validates the loss of performed to assess whether these mice exhibit changes in hα-Syn α-andβ-Syn expression in α-Syn–KO, β-Syn–KO, and triple Syn–KO neurons +/+ aggregation and solubility, compared to hα-Syn controls. In line compared to WT neurons. (B) Significantly more β-Syn–KO neurons and with our previous data, less monomeric hα-Syn was detected in cy- triple-KO neurons (one-way ANOVA and Scheffé post hoc analysis) develop +/+ −/− > μ ± = tosolic fractions derived from hα-Syn SNCA mice than in cy- inclusions ( 1 m in diameter) compared to non-Tg neurons (mean SD, n hα-Syn+/+ A 3 independent experiments, 20 neurons per condition). (C) Inclusions formed tosolic fractions derived from counterparts (Fig. 6 ). This in β-Syn–KO and triple Syn–KO neurons are pS129–α-Syn–positive. Colocali- decrease in monomeric hα-Syn was concomitant with the appear- zation was confirmed by assessing regression coefficients (R2). (Scale bars: ance of HMW hα-Syn species within particulate fractions, which 20 μminA and 5 μminB and C.)

Fares et al. PNAS | Published online February 2, 2016 | E917 Downloaded by guest on September 30, 2021 with multimeric/aggregated hα-Syn species rather than hα-Syn monomers, we assessed the interaction of mα-Syn monomers with equimolar amounts of hα-Syn monomers or hα-Syn PFFs (characterized in Fig. S8). Intriguingly, mα-Syn was coimmunoprecipitated only when it was mixed with hα-Syn PFFs and not with hα-Syn monomers (Fig. 7A), indicating that monomeric mα-Syn might in- teract preferentially with hα-Syn PFFs in vitro. To confirm these findings and to obtain a quantitative assessment of the interaction between mα-Syn monomers and hα-Syn PFFs by an independent readout, we performed SPR measurements. Briefly, saturating amounts of either hα-Syn monomers or hα-Syn PFFs were immobilized by amine coupling to separate CM5 chips, and then increasing concentrations of mα-Syn monomers were flushed onto the chips. As shown in Fig. 7B, and in line with our immunoprecipitation experiments,nointeractionwasnotedbetweeninjectedmα-Syn monomers and immobilized hα-Syn monomers, even at the highest tested concentration for mα-Syn (10 μM). In contrast, mα-Syn monomers interacted readily with sonicated hα-Syn PFFs in a dose-dependent manner, even at concentrations as low as 0.1 μM. To investigate whether endogenous mα-Syn similarly interacts with aggregated hα-Syn species in primary neurons, we treated WT neurons with sonicated hα-Syn PFFs and assessed interaction by immunoprecipitation and immunofluorescence analyses. Impor- tantly, and in line with our in vitro results, mα-Syn coimmunoprecipitated with pulled-down hα-Syn PFFs (Fig. S7B). Moreover, confocal imaging revealed that internalized hα-Syn PFFs colocalize with endogenous mα-Syn (Fig. S7C), which redistributes from a diffuse somatic localization into defined puncta as previously described (22). Together, these results show that mα-Syn interacts preferentially with aggregated hα-Syn PFFs in vitro and in neurons. Next, we explored whether such an interaction between α-Syn PFFs and α-Syn monomers from different species would seed or, alternatively, attenuate progressive monomer aggregation. To do so, we assessed the aggregation kinetics of hα-Syn or mα-Syn monomers in the presence of either hα-Syn PFFs or mα-Syn PFFs (characterized in Fig. S8). Strikingly, assessment of thioflavin-T (ThT) binding and the formation of fibrillar structures by TEM Fig. 6. The absence of mα-Syn promotes hα-Syn aggregation in transgenic showed that the seeding efficiency is significantly higher when hα- mice. (A) Significantly less monomeric hα-Syn is detected in cytosolic frac- Syn monomers and PFFs are of the same species (Fig. 7C and Fig. tions from hα-Syn+/+SNCA−/− mice than in hα-Syn+/+ counterparts (n = 3in- S9 A and B). For instance, whereas the lag phase was eliminated in dependent experiments), concomitant with the appearance of HMW hα-Syn the mixture of hα-Syn monomers and hα-Syn PFFs, resulting in the −/− species in particulate fractions. Loss of mα-Syn expression in SNCA mice appearance of fibrillar structures at 8 h of incubation, the aggre- α – was verified using the m -Syn specific antibody D37A6, and actin was used gation profile of the mixture of hα-Syn monomers and mα-Syn to control for equal protein loading. The asterisk indicates a nonspecific PFFswassimilartothatofhα-Syn alone, in which only oligomeric band in cytosolic fractions. (B) Significantly more proteinase K (PK)-resistant hα-Syn inclusions/0.1 mm2 are detected in the neocortex and hippocampus species were formed at 8 h of incubation. Interestingly, the mixture + + − − + + α α (Hipp.) of hα-Syn / SNCA / mice than in hα-Syn / mice (n = 3 mice per of m -Syn monomers with m -Syn PFFs showed the most dramatic condition). (Scale bars: 100 μm in overviews and 15 μm in insets.) In A and B elimination of the lag phase, with fibrils formed after only 2 h of the Mann–Whitney test was applied to obtain P values. (C and D) Double- incubation. Here again, although the addition of hα-Syn PFFs to positive hα-Syn inclusions for total α-Syn and pS129 α-Syn (C) or synapto- mα-Syn monomers resulted in higher ThT plateau values than seen physin (D) are detected in the cortex, striatum, and hippocampus of with mα-Syn monomers alone, it failed to eliminate the lag phase +/+ −/− hα-Syn SNCA mice. Orthogonal (Orth.) Z-stack projections verify the and resulted in the formation of oligomeric species at 2 h. Taken μ intracellular nature of inclusions. (Scale bars: 5 m.) together, these findings strongly show that the aggregation kinetics of α-Syn are significantly accelerated when the seeds and monomers are from the same species, and that the presence of mixed addition, immunohistochemical analysis showed that hβ-Syn was species of monomers and PFFs results in reduced seeding capacity localized mostly within neuronal terminals and did not form any +/+ and slower aggregation kinetics. inclusions in the brains of hβ-Syn mice, as previously reported α hβ-Syn+/+SNCA−/− F To evaluate whether the seeding and spreading potential of -Syn (37), or in the brains of mice (Fig. S6 ). PFFs is similarly affected by the presence of PFFs and endogenous α These results show that -Syn ablation does not promote the monomers of the same species in the brain, we injected mα-Syn β −/− aggregation of h -Syn in vivo. PFFs or hα-Syn PFFs into the striatum of WT (non-Tg) or SNCA mice (as controls) and then compared the seeding and spreading α α m -Syn Interacts with Aggregated h -Syn Species and Attenuates propensity in different brain regions 1 month postinjection. Strik- α Seeding and Spreading. To investigate the mechanism by which m - ingly, we observed the most pronounced spreading of α-Syn pa- Syn affects hα-Syn aggregation, we first assessed whether overex- thology only when mα-Syn PFFs were injected into WT mice pressed hα-Syn interacts directly with endogenous mα-Syn in WT expressing endogenous mα-Syn (Fig. 7D); in these mice mα-Syn primary neurons. Notably, we did not detect any interaction be- accumulated into somatic inclusions that were ubiquitinated tween the two proteins at the monomer level by coimmunopre- and hyperphosphorylated at S129 in the cortex and amygdala cipitation (Fig. S7A). To investigate whether mα-Syn interacts (Fig. S9C). In contrast, hα-Syn PFFs induced significantly less

E918 | www.pnas.org/cgi/doi/10.1073/pnas.1512876113 Fares et al. Downloaded by guest on September 30, 2021 PNAS PLUS

Fig. 7. mα-Syn interacts with aggregated hα-Syn species and attenuates seeding and spreading. (A) Following immunoprecipitation (IP) of hα-Syn, mα-Syn monomers (M) were detected only in the sample comprising the mixture of mα-Syn M with hα-Syn PFFs, and not with hα-Syn M. The asterisks indicate the NEUROSCIENCE heavy and light chains of the antibody used for IP. (B) A dose-dependent response (interaction) was noted by SPR between immobilized hα-Syn PFFs and injected mα-Syn M, but not between the injected mα-Syn M and immobilized hα-Syn M. (C) Mixtures of α-Syn PFFs and monomers of the same species show significantly higher ThT binding at early time points than seen with α-Syn monomers alone or with PFFs of different species (n = 3 independent experiments). Mean ± SD values are shown. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA and Scheffé post hoc analysis). (D) Significantly more pS129–α-Syn inclusions (arrows) are observed in the cingulate cortex and amygdala of WT mice injected with mα-Syn PFFs (n = 5 mice per condition) than in counterparts − − injected with hα-Syn PFFs or in SNCA / mice injected with hα-Syn/mα-Syn PFFs, which show diffuse staining (arrowheads). The Mann–Whitney test was applied to obtain P values. (Scale bar: 100 μm.)

pathology at these regions in WT mice at the same time point, solely on the overexpression of hα-Syn have reported inlcusions that −/− and SNCA mice showed very limited overall pathology following exhibit these features altogether. injection with either hα-Syn PFFs or mα-Syn PFFs. These Multiple lines of evidence from many of our experiments further −/− findings are consistent with the significantly reduced seeding converged to establish that the formation of inclusions in SNCA efficiency of hα-Syn PFFs in the presence of monomeric mα- neurons is an aggregation-driven process. Overexpression of the Syn in vitro; this reduced seeding efficiency may allow more aggregation-incompetent mutant S87E or treatment with the α time for natural inhibitory or clearance mechanisms to interfere pharmacological -Syn aggregation inhibitor tolcapone significantly with the seeding and spreading of exogenous hα-Syn PFFs in reduced the formation of inclusions. In contrast, overexpression of α mouse brains. the aggregation-prone truncated h -Syn variant (1-120) resulted in the formation of larger inclusions than seen with the WT protein. Discussion In all cases in which we observed increased inclusion formation, we α The aggregation of α-Syn into LBs has been directly linked to the also observed decreased h -Syn solubility and the appearance of pathogenesis of PD (2). The mechanism of formation and the HMW aggregates, concomitant with the loss of monomers. More- precise effect of LBs on neuronal physiology and viability re- over, time-lapse live imaging coupled with FRAP and photoconversion experiments showed in real time that the inclusions grow mains poorly understood, mostly because of the lack of animal α and cellular models exhibiting de novo α-Syn fibrillization into in size and readily incorporate soluble h -Syn protein. Interestingly however, when we explored the toxic potential of these inclusions, LB-like structures. Although doubling α-Syn expression is suffi- we found that the aggregates formed do not provoke increased cient to cause PD in humans, attempts to reproduce molecular toxicity (SI Discussion and Fig. S10). This finding is consistent with PD pathology based on α-Syn overexpression in various animal previous studies suggesting that intracellular hα-Syn aggregates models have not been successful. This lack of success could imply α may be protective in cellular models (38, 39). Further studies are the presence of cellular factors that inhibit h -Syn aggregation in underway to determine whether these aggregates exhibit a similar these models. In this study, we sought to systematically examine role under conditions of cellular stress, such as treatment with α α whether endogenous -Syn homologs attenuate h -Syn aggregation mediators of oxidative stress or inhibitors of protein degradation, SNCA−/− in mouse neurons. We found that a substantial fraction of or other molecules known to contribute to PD pathogenesis. − − neurons expressing hα-Syn develop inclusions that exhibit key LB- Having established that SNCA / primary neurons are more like features, including S129 hyperphosphorylation, ubiquitination, permissive of hα-Syn aggregation in culture, we sought to validate and ThS reactivity. Importantly, this phenomenon reflected de novo this finding in vivo. Two previous independent studies had suggested Δ hα-Syn fibrillization, as revealed by ssTEM following both CLEM that the expression of hα-Syn 120 or hα-SynA53T in Tg mice lacking and immunogold-labeling experiments. To the best of our knowl- mα-Syn expression leads to the formation of fibrillar inclusions edge, none of the previously published cellular models that rely within the substantia nigra or spinal cord (5, 6). Therefore, we

Fares et al. PNAS | Published online February 2, 2016 | E919 Downloaded by guest on September 30, 2021 directly compared the aggregation propensity of hα-Syn in brains of aggregation. Once this level drops below a certain threshold (in mice expressing or lacking endogenous mα-Syn. Our data showed either single or triple KOs), the aggregation of hα-Syn into LB-like that hα-Syn exhibits enhanced aggregation propensity in the absence inclusions becomes progressive. Supporting this proposition is of mα-Syn, as reflected by decreased solubility, enhanced detection the observation that intermediate reduction of mα-Syn levels by of HMW oligomeric species, and the formation of abundant pro- shRNA did not promote the formation of hα-Syn inclusions in teinase K-resistant and pS129 hyperphosphorylated inclusions. To WT neurons. rule out the possibility that knocking out mα-Syn promotes non- In summary, we have shown that two endogenous members of specific aggregation, we conducted similar studies on hβ-Syn, the the synuclein family, mα-Syn and mβ-Syn, attenuate the aggrega- α-Syn homolog that is significantly less prone to aggregate. As tion of overexpressed hα-Syn in mouse neurons. Taken together expected, knocking out mα-Syn did not affect the levels, distribution, with the multitude of studies that have reported the inhibition of or staining pattern of hβ-Syn in Tg mice. − − aggregation of other amyloidogenic proteins by their homologues Our observation that the reintroduction of mα-Syn in SNCA / (such as Aβ, TTR, hemoglobin, and IAPP), this observation could neurons significantly attenuates the formation of hα-Syn inclusions reflect a more generic biological phenomenon in which endoge- suggests that the mouse homolog might directly inhibit hα-Syn ag- nous homologs act as natural inhibitors of abnormal aggregation. gregation. Intriguingly, although we could not detect a prominent This phenomenon could explain why most current rodent PD interaction between hα-Syn and mα-Syn at the monomeric level in models relying on hα-Syn overexpression in WT mice do not show neurons, we consistently observed that monomeric mα-Syn prefer- prominent hα-Syn fibrillization. As such, understanding the in- entiallyinteractswithsonicatedPFFsinvitroandinneurons. teractions between homologous proteins and the effect of such Moreover, we found that this interaction does not promote pro- interactions on protein aggregation and amyloid formation could gressive seeding and spreading of pathology in vitro or in mouse facilitate the generation of novel cellular and in vivo models of brains in vivo. As such, endogenous mα-Syn might directly inhibit protein misfolding. The ability to assess the dynamics of inclusion α h -Syn fibrillization in WT primary neurons by stabilizing oligo- formation quantitatively in our neuronal model provides unique meric species and capping the terminals of early fibrillar structures opportunities to elucidate molecular and cellular determinants α formed by h -Syn. This hypothesis is in line with the early study by that influence the mechanisms of α-Syn fibrillization in living α Rochet et al. (7) showing that m -Syn inhibits the fibrillization of neurons, and to identify pathways that modulate this process and α h -Syn by stabilizing oligomers on the pathway to amyloid forma- contribute to neurodegeneration. Further studies using this model tion in vitro. Moreover, it is consistent with literature reports could make it possible to identify novel drugs for the treatment of α demonstrating that the propagation and spreading of -Syn pa- PD based on modulating α-Syn aggregation and interneuronal thology is prominent when the injected PFFs and host-expressed spreading, or enhancing the degradation and clearance of toxic monomers are from the same species. In most of these studies, α-Syn aggregates. seeding and spreading of α-Syn pathology was observed when mα- Syn PFFs were injected into non-Tg mice expressing mα-Syn (40– Materials and Methods α 42)orwhenh-Syn PFFs were injected into Tg mice expressing Full experimental details are provided in SI Materials and Methods. human α-Syn protein (43–48). An attractive general implication of our findings is that the Approval of Animal Experimentation. All animal experiments were performed attenuation of aggregation by homologous proteins could represent in accordance with guidelines and regulations of the Ecole Polytechnique a generic biological phenomenon. This proposition is consistent Fédérale de Lausanne (EPFL), Cardiff University, and University of California with multiple studies reporting decreased aggregation kinetics in the San Diego, and were approved by the Swiss Federal Veterinary Office. presence of homologous amyloidogenic proteins underlying different diseases. For instance, the key peptides involved in the forma- Primary Neuron Culture Preparation, Transfection, Infection, and Tolcapone tion of amyloid plaques in Alzheimer’s disease, Aβ40 and Aβ42, Treatment. Primary hippocampal and cortical neuronal cultures were pre- were shown to inhibit each other’s aggregation reciprocally in vitro pared from postnatal day 0 C57BL/6JRccHsd (WT; Harlan Laboratories), α × tm1Rosl in a concentration-dependent manner (49). Moreover, mutant he- C57BL/6JOlaHsd ( -Syn KO; Harlan Laboratories) (12), B6,129 1-Snca /J β (α-Syn–KO; Jackson Laboratories) (13), β-Syn–KO (31), γ-Syn–KO (58), or moglobin -chain polymerization in sickle cell anemia was shown to α β γ – γ - - -Syn KO (33) mice as previously described (10). Briefly, dissected tissues be attenuated upon increasing ratios of its -chain homolog (50). were dissociated with papain and triturated using a glass pipette. After Likewise, in transthyretin (TTR) amyloid diseases, murine/human centrifugation at 400 × g for 2 min, cells were plated in minimum essential TTR homologs form heterotetramers that impair amyloid forma- medium (MEM)/10% (vol/vol) horse serum onto poly-L-lysine (Sigma)–coated tion (51, 52), and aggregation in vivo was achieved mostly in Tg coverslips [12 mm glass (VWR International) for immunocytochemistry or mice expressing TTR mutants on an endogenous TTR-KO back- 13 mm plastic (Thermanox, Nunc) for immunogold staining and TEM] at 1.5 × ground (52). Finally, the aggregation kinetics of the islet amyloid 105 cells/mL, or at 3 × 105 cells/mL in 35-mm confocal dishes (World Precision) polypeptide (IAPP) in bulk solution was reduced dramatically by for live imaging, or in 10-cm dishes (BD Biosciences) for biochemical analysis. the presence of its rat homolog (53, 54). Medium was changed after 4 h to Neurobasal/ B27 medium, and neurons In light of these findings, we investigated whether this phe- were treated with Arabinosylcytosine (AraC; Sigma) after 6 d in vitro (DIV) to nomenon could be extended to the other endogenous hα-Syn ho- stop glial division. At 7 DIV, neurons were either transiently transfected for β γ β – up to 3 d using Lipofectamine 2000 (Invitrogen) according to the manu- mologs, -Syn and -Syn. Interestingly, we found that -Syn KO ’ α facturer s instructions or were infected with lentiviruses at a multiplicity of cultures also developed h -Syn inclusions that were hyper- infection (MOI) of 10 for up to 7 d. When indicated, at 24 h posttransfection, phosphorylated at S129. In contrast, the distribution of hα-Syn neurons were treated with 20 μM tolcapone (Sigma) for an additional was not affected when expressed in γ-Syn–KO neurons, be- 24 h before immunocytochemistry. cause the levels of γ-Syn in WT hippocampal neurons were already very low and therefore could not be reduced drasti- ACKNOWLEDGMENTS. We thank Nathalie Jordan and Celine Vocat for help cally in γ-Syn–KO neurons. These results are also in line with in preparing primary cultures, plasmid cloning, and production of recombi- β α nant proteins; Mr. Samuel Kilchenmann for expert help in conducting and previous reports showing that -Syn inhibits -Syn aggregation analyzing surface plasmon resonance (SPR) experiments; Dr. Anne-Laure in vitro (55, 56) as well as in vivo; double-Tg mice coex- Mahul and Dr. Issa Issac for helping with blinded quantification of some pressing both hα-Syn and hβ-Syn exhibit ameliorated motor experiments; Mr. Anass Chiki for help with protein characterization; Mr. Maroun deficits and lower α-Syn accumulation compared to Tg mice Bou Sleiman for help with statistical analysis; Prof. Virginia Lee for kindly pro- α viding the Syn-208 antibody; and Mrs. Dania Al Labban and Dr. Juan Reyes for expressing h -Syn alone (37, 57). However, our results fur- their critical evaluation of the manuscript. This work was supported by the Ecole thersuggestthatthetotallevels or ratio of both endogenous Polytechnique Fédérale de Lausanne (EPFL), the Swiss National Science Foun- α-andβ-Syn could be critical in protecting against hα-Syn dation, and Wellcome Trust Grant 075615/Z/04/z (to V.L.B.).

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