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Propagation of pathological α-synuclein in marmoset brain

Propagation of pathological α-synuclein in marmoset brain α-Synuclein is a defining, key component of Lewy bodies and Lewy neurites in Parkinson’s disease (PD) and dementia with Lewy bodies (DLB), as well as glial cytoplasmic inclusions in multiple system atrophy (MSA). The distribution and spreading of these pathologies are closely correlated with disease progression. Recent studies have revealed that intracerebral injection of synthetic α-synuclein fibrils or pathological α-synuclein prepared from DLB or MSA brains into wild-type or transgenic animal brains induced prion-like propagation of phosphorylated α-synuclein pathology. The common marmoset is a very small primate that is expected to be a useful model of human diseases. Here, we show that intracerebral injection of synthetic α-synuclein fibrils into adult wild-type marmoset brains (caudate nucleus and/or putamen) resulted in spreading of abundant α-synuclein pathologies, which were positive for various antibodies to α-synuclein, including phospho Ser129-specific antibody, anti-ubiquitin and anti-p62 antibodies, at three months after injection. Remarkably, robust Lewy body-like inclusions were formed in tyrosine hydroxylase (TH)-positive neurons in these marmosets, strongly suggesting the retrograde spreading of abnormal α-synuclein from striatum to substantia nigra. Moreover, a significant decrease in the numbers of TH-positive neurons was observed in the injection-side of the brain, where α-synuclein inclusions were deposited. Furthermore, most of the α-synuclein inclusions were positive for 1-fluoro-2,5-bis (3-carboxy-4-hydroxystyryl) benzene (FSB) and thioflavin-S, which are dyes widely used to visualize the presence of amyloid. Thus, injection of synthetic α-synuclein fibrils into brains of non-transgenic primates induced PD-like α-synuclein pathologies within only 3 months after injection. Finally, we provide evidence indicating that neurons with abnormal α-synuclein inclusions may be cleared by microglial cells. This is the first marmoset model for α-synuclein propagation. It should be helpful in studies to elucidate mechanisms of disease progression and in development and evaluation of disease-modifying drugs for α-synucleinopathies. Keywords: α-synuclein, Parkinson, Prion, Marmoset, Circuits Introduction component of glial cytoplasmic inclusions (GCIs) in mul- Parkinson’s disease (PD) is the second most common tiple system atrophy (MSA) [54, 58]. These diseases are neurodegenerative disease after Alzheimer’s disease, and collectively referred to as α-synucleinopathies. To date, six Lewy bodies (LBs) and Lewy neurites (LNs) are character- missense mutations in the SNCA gene and occurrence of istic features of PD. Dementia with Lewy bodies (DLB) is gene multiplication have been identified in familial forms also a progressive neurodegenerative disease characterized of PD and DLB [1, 5, 24, 28, 29, 41, 52, 62]. α-Synuclein is by the appearance of LBs and LNs in cortex [17, 22]. The a small protein of 140 amino acids, which is localized in discovery of disease-associated mutation in the α-synuclein presynaptic termini, and is involved in maintenance of gene SNCA and subsequent immunostaining studies with synapses and synaptic plasticity. In PD, DLB, or MSA pa- antibodies demonstrated that α-synuclein is the major tients, it is deposited in the brain as a filamentous form component of LBs and LNs [2, 55, 56]. It is also the major with cross-β structure [51], which is abnormally phos- phorylated at Ser129 and partially ubiquitinated [15, 21]. α-Synuclein is natively unfolded, but readily assembles * Correspondence: hasegawa-ms@igakuken.or.jp into amyloid-like fibrils under appropriate conditions. Department of Dementia and Higher Brain Function, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506, Japan Pathogenic mutations affect fibril formation in vitro, either Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Shimozawa et al. Acta Neuropathologica Communications (2017) 5:12 Page 2 of 14 accelerating fibril formation [6, 7, 16] or resulting in were originally one structure and that they became sepa- formation of fibrils that are more fragile and easier to rated by the internal capsule during evolution [25]. propagate than wild-type (WT) fibrils [61]. Moreover, Thus, the marmoset has advantageous characteristics as the spreading of pathological α-synuclein is closely corre- an experimental animal to study brain networks, func- lated with disease progression; indeed, the distribution tions and disease conditions. pattern and spread of the pathologies are useful for disease Here, we investigated whether intracerebral injection of staging of sporadic PD [3, 48]. These results suggest that α-synuclein fibrils can induce PD/DLB-like pathologies in intracellular amyloid-like α-synuclein fibrils can cause PD marmoset, and we present the first marmoset model of and DLB, and spreading of α-synuclein pathology in the α-synuclein propagation. We found that marmosets devel- brain is considered to be the underlying mechanism of oped abundant phosphorylated α-synuclein pathologies, progression of these diseases. Recently, it was experi- similar to those observed in PD/DLB, in various brain mentally demonstrated that intracerebral injection of regions, including striatum, cortex and substantia nigra, at synthetic α-synuclein fibrils and/or insoluble α-synuclein only three months after injection. Remarkably, many LB- from diseased brain converts normal α-synuclein into an like inclusions are observed in tyrosine hydroxylase (TH)- abnormal form, and the abnormal α-synuclein propagates positive dopamine neurons, and a significant decrease in throughout the brain in a prion-like manner in WT TH-staining was seen in the injection hemisphere. The mouse [30, 33, 34, 57], α-synuclein transgenic mouse inclusions were also positive for fluorescent β-sheet ligands, [31, 36, 60] and monkey [44]. thioflavin-S and FSB, implying that α-synuclein deposits in Common marmoset (Callithrix jacchus) is a very small these animals should be detectable in vivo by positron new world primate, about 25 – 35 cm in height and emission tomography (PET) with a suitable small- 300 – 500 g in weight, and is far more experimentally molecular agent. Taking account of the advantages of mar- tractable than macaque monkey. Since it has high fecund- mosets over mice, we believe the current experimental ity, with a short sexual maturation period of 18 months, it model would be particularly useful to examine the relation- is attracting increasing attention as an experimental model ships between PET-detectable α-synuclein lesions and dis- of primates. In fact, a national project called Brain/ ruptions of neural networks in the absence and presence of MINDS (Brain Mapping by Integrated Neurotechnologies candidate α-synucleinopathy-modifying therapeutics. for Disease Studies) was started in 2014 in Japan to de- velop the common marmoset as a model animal for Materials and methods neuroscience [19, 38, 39]. The marmoset cortex is rela- Preparation of recombinant α-synuclein and fibrils tively smooth, but the gyrencephalic and cortical sheet is Recombinant human and mouse wild-type α-synuclein divided into functionally distinct cortical areas, as in Old and fibrils were prepared as described previously [33, 57]. World monkeys [45], and thus is suitable for studies of Briefly, purified α-synuclein (7 – 10 mg/ml) was incubated higher cognitive functions and social communication [11]. at 37 °C in a shaking incubator at 200 rpm in 30 mM Therefore, marmosets are considered to be a good experi- Tris–HCl, pH 7.5, containing 0.1% NaN , for 72 h. α- mental model animal to understand the evolution of brain Synuclein fibrils were pelleted by spinning the assembly development and function. Moreover, transgenic marmo- mixtures at 113,000 xg for 20 min, resuspended in 30 mM sets have already been generated, demonstrating the feasi- Tris–HCl buffer (pH 7.5), and sonicated for 3 min (Biomic bility of gene manipulation in this species [49]. 7040 Ultrasonic Processor, Seiko). The protein concentra- To date, mouse models have been used to investigate tions were determined by HPLC. Samples were run on brain development, circuits, and higher cognitive func- gradient 12% polyacrylamide gels and stained with Coo- tions, but they have limitations for exploration of the massie Brilliant Blue (CBB), or electrophoretically trans- evolution and development of the primate neocortex. In ferred to PVDF membranes. For immunoblotting, situ hybridization analysis of marmoset brain revealed membranes were incubated with 3% gelatin (Wako) for that the expression patterns of the genes that regulate 10 min at 37 °C, followed by overnight incubation at room brain development (such as EphA6) are different, especially temperature with primary antibodies. Next, the mem- in brain areas that have connections to the prefrontal branes were incubated for 1 hr at room temperature with cortex and are presumably involved in higher cognitive biotinylated anti-rabbit or mouse IgG (Vector Lab), then functions, although similar broad regional patterns of incubated for 30 min with avidin-horseradish peroxidase expression were observed in both species [32]. (Vector Lab), and the reaction product was visualized by A particular difference in brain development and using 0.1% 3,3-diaminobenzidine (DAB) and 0.2 mg/ml structure between mouse and marmoset is that striatum NiCl as the chromogen. For electron microscopy, sam- of marmoset is separated into caudate nucleus and puta- ples were placed on collodion-coated 300-mesh copper men, while these are not distinguishable in rodents. It grids, stained with 2% (v/v) phosphotungstate, and exam- has been considered that caudate nucleus and putamen ined with a JEOL 1200EX electron microscope. Shimozawa et al. Acta Neuropathologica Communications (2017) 5:12 Page 3 of 14 Marmosets detection and characterization of α-synuclein pathologies According to animal protection considerations based on in marmoset brains. Anti-p62 (Progen), anti-Ub (Dako, the 3R (reduce, reuse, recycle) principle, we designed the Millipore), anti-TH (Millipore), anti-NeuN (Millipore), experiment very carefully to minimize the number of anti-GFAP (Sigma), anti-CNPase (Abcam) and anti-Iba1 animals used. Two female 26-month-old marmosets (Wako) antibodies were also used. (individual recognition No. 14H and 14I; born on 4th April, 2014 and bred at the Animal Research Division, Tokyo Metropolitan Institute of Medical Science) were Immunohistochemistry used for this experiment. Marmosets were deeply anesthetized with pentobarbital injection and killed, and the brain was perfused with Stereotaxic surgery 0.1 M phosphate buffer, followed by 10% formalin neutral The marmosets were anesthetized with Ketamine Hydro- buffer solution. After fixation, whole brains were sectioned chloride (20–40 mg/kg i.m.) and Xylazine (0.05 mg/kg coronally at 50 μm using a vibratome (Leica, Wetzlar, i.m.), and Butorphanol (0.05–0.1 mg/kg i.m.). Then, 50 μL Germany). For high-sensitivity detection, free-floating aliquots of 4 mg/mL mouse α-synuclein fibrils were brain sections were treated with formic acid for 20 min, injected into both caudate nucleus (interaural +9.5 mm, washed, and boiled at 100 °C for 20 min as described [33]. Lateral 3 mm, Depth 6 mm) and putamen (interaural Sections were then incubated with 0.5% H O in methanol 2 2 +9.5 mm, Lateral 6 mm, Depth 3 mm) in the right hemi- for 30 min to inactivate endogenous peroxidases, blocked sphere of 14H brain (total 400 μg). A 50 μL aliquot was with 10% calf serum in PBS for 20 min, and incubated injected into caudate nucleus (interaural +9.5 mm, Lateral overnight with appropriate antibodies. After incubation 3 mm, Depth 6 mm) in the right hemisphere of 14I brain with the biotinylated secondary antibody for 2 h, labeling (total 200 μg). The marmosets were bred for 3 months was detected using the ABC staining kit (Vector) with after injection in a biological safety level 2 (BSL-2) envir- DAB. Sections were counterstained with hematoxylin. onment. All experimental protocols were approved by the Slides were coverslipped with mounting medium. Images Animal Care and Use Committee of Tokyo Metropolitan were observed with an all-in-one microscope/digital Institute of Medical Science (No. 16038). camera (BZ-X710; Keyence). For double-label immunofluorescence detection, brain Antibodies sections were pretreated as described above and incubated Primary antibodies used in this study are listed in Table 1. overnight at 4 °C with a cocktail of appropriate primary An anti-phosphorylated α-synuclein rabbit monoclonal antibodies. The sections were washed and incubated with antibody to pS129 (Abcam) and other anti-α-synuclein a cocktail of Alexa568-conjugated goat anti-mouse or antibodies, including LB509 [26] (a gift from Dr anti-rabbit IgG and Alexa488-conjugated goat anti-mouse Iwatsubo), 75–91 (Cosmo bio), 131–140 (Cosmo bio) or anti-rabbit or anti-guinea pig IgG (Molecular Probes). and #2642 (Cell Signaling Technology) were used for After further washing, the sections were coverslipped with Table 1 Antibodies used in this study Primary antibodies Type Source Dilution pS129 (phosphorylated a-syn) rabbit mono Abcam (ab51253) 1:2000 LB509 (human a-syn) mouse mono Gift from Dr Iwatsubo 1:1000 75–91 (a-syn 75–91) rabbit poly Cosmo bio (CAC-TIPSNP08) 1:1000 131–141 (a-syn 131–140) rabbit poly Cosmo bio (CAC-TIPSNP09) 1:1000 #2642 (a-syn) rabbit poly Cell Signaling Tech (#2642) 1:1000 Anti-p62 guinea pig poly Progen (GP62-C) 1:1000 Anti-Ub rabbit poly Dako (Z0458) 1:1000 Anti-Ub mouse mono Millipore (MAB1510) 1:1000 Anti-TH rabbit poly Millipore (AB152) 1:1000 Anti-TH mouse mono Millipore (MAB318) 1:1000 Anti-NeuN mouse mono Millipore (MAB377) 1:1000 Anti-GFAP mouse mono Sigma (G3893) 1:1000 Anti-CNPase mouse mono Abcam (ab6319) 1:200 Anti-Iba1 rabbit poly Wako (016–20001) 1:1000 Shimozawa et al. Acta Neuropathologica Communications (2017) 5:12 Page 4 of 14 non-fluorescent mounting media (VECTASHIELD; mouse α-synuclein fibrils instead of human α-synuclein Vector Laboratories) and observed with the BZ-X710. fibrils for injection into marmoset brains in this experi- To measure positive cells, 7–9 sections of substantia ment. This had the advantage that we could investigate nigra were randomly selected, and all images were cap- whether endogenous marmoset α-synuclein is deposited tured with BZ-X710 microscope using the same settings. in brains injected with mouse α-synuclein fibrils, be- The areas of pS129-positive cells and TH-positive cells cause the two can be distinguished with antibodies such in the right and left substantia nigra were extracted and as LB509. Recombinant mouse α-synuclein and the fibrils quantified by BZ-H3C Hybrid Cell Count Software were prepared as described, and images of purified recom- (Keyence). binant mouse α-synuclein and the fibrils are shown in Fig 2. EM pictures of the injected sample showed that Thioflavin-S and FSB stainings most of the fibrils were straight, 5 – 10 nm in width, and Thioflavin-S and FSB [23, 50] were purchased from 50 – 300 nm in length (Fig 2C). The prion-like seeding Sigma-Algrich and Dojindo, respectively. For fluorescence activity of the fibrils to convert normal α-synuclein into labeling with β-sheet ligands, thioflavin-S and FSB, brain abnormal form was checked in our cultured cell model sections were mounted on a glass slide and dried with [37] and mouse model [57] (data not shown). We injected warm air. Sections were incubated in 20% ethanol con- 200– 400 μgof the α-synuclein fibrils; this amount was taining 0.001% β-sheet ligands at room temperature for chosen based on the amount used in the previous mouse 30 min. The samples were rinsed with 20% ethanol for experiments (10 μg fibrils/0.3 g mouse brain vs 200 μg 5 min, dipped into distilled water twice for 3 min each, fibrils/7 g marmoset brain). and mounted in VECTASHIELD. Fluorescence images were captured using a BZ-X710 fluorescence microscope PD-like pathologies in marmoset brain within only equipped with Filter set ET-ECFP (Chroma Technology) 3 months after injection for thioflavin-S and FSB. After fluorescence microscopy, Three months after injection, formalin-fixed marmoset all sections labeled with ligands were autoclaved for anti- brain sections were prepared and immunostained with gen retrieval, immunostained with the pS129 antibody, anti-phospho-Ser129 of α-synuclein (pS129). Surprisingly, and examined using the BZ-X710 instrument. abundant pS129-positive structures were observed throughout the brain regions, including the injection Results sites in the caudate nucleus and putamen, accumbens, Inoculation of mouse α-synuclein fibrils into marmoset brain bed nucleus of the stria terminalis, substantia nigra, amyg- Marmoset α-synuclein, mouse α-synuclein and human dala, a wide region of cortex, thalamus, globus pallidus, α-synuclein share 96 – 97% amino acid sequence hom- dorsal raphe nuclei, raphe nucleus and hippocampal CA1 ology (Fig 1), but marmoset and mouse α-synuclein pro- in both marmosets (Fig 3). These abnormal α-synuclein teins both have a threonine residue at amino acid pathologies were also positive with other anti-α-synuclein position 53, which is an aggregation-prone mutation in antibodies (Fig 4), including LB509. The results indicated familial Parkinson’s disease [41]. Therefore, we used that endogenous marmoset α-synuclein is converted into Fig. 1 Comparison of amino acid sequences of human (Homo sapiens), marmoset (Callithrix jacchus)and mouse (Mus musculus) α-synuclein. Amino acids in human α-synuclein that differ from those of marmoset and mouse α-synuclein are indicated in red, while amino acids in marmoset and mouse α-synuclein that differ from those in human α-synuclein are indicated in green and blue, respectively. Epitopes of α-synuclein antibodies used in this study are also indicated Shimozawa et al. Acta Neuropathologica Communications (2017) 5:12 Page 5 of 14 Fig. 2 Characterization of purified mouse α-synuclein and fibrils. a, purified monomeric human α-synuclein (lane 1), mouse α-synuclein (lane 2) and mouse α-synuclein fibril (lane 3) samples were analyzed by CBB staining (0.5 μgofprotein/lane). b, results of immunoblotting with anti-α-synuclein antibodies (131–140 and LB509) (B, 0.01 μgofprotein/lane). c, electron microscopy of α-synuclein fibrils injected into marmoset brains in this study. Negatively stained short fibrils, 50 –300 nm in length, were observed. Scale bar: 100 nm an abnormal form by inoculation of mouse α-synuclein fi- regions over the 3-month period. Overall, the spreading brils and deposited in the brain. Most of the inclusions pattern of pathological α-synuclein seemed to be the were also positive for antibodies to ubiquitin and p62 same as in mouse brain [30, 33, 57], and was consistent (Fig 5), as is the case in the brains of patients. Labeling of with the spreading pattern of retrograde tracers [40]. the inclusions with these antibodies was observed without It is noteworthy that abundant LB-like round pS129- pretreatment of sections with formic acid and boiling, but positive inclusions were detected in substantia nigra of the specificity and sensitivity were both increased by the these marmosets (Fig 3, 4, 5 and 7). The nigral LB-like pretreatment (data not shown). inclusions were more prominent in the marmoset injected into both caudate nucleus and putamen than in the α-Synuclein propagated retrogradely through neuronal marmoset injected only into caudate nucleus. Double networks labeling of the inclusions with anti-tyrosine-hydroxylase The distributions of the pathologies were similar in these (TH) antibody confirmed that the inclusions are formed in two marmosets 14H and 14I, although more abundant TH-positive dopaminergic neurons (Fig 7b, c), indicating and severe pathologies were observed in the marmoset that pathological α-synuclein was propagated retrogradely 14H (Fig 6), in which α-synuclein fibrils were injected at from striatum to nigral neurons. both caudate nucleus and putamen. The α-synuclein pathologies were robust in the injected right hemisphere, but also present in the left hemisphere, although to a Degeneration of dopaminergic neurons with pS129- much lesser extent than in the right hemisphere (Fig 6). positive inclusions The spreading of α-synuclein pathology observed in the Macroscopical observation of sections stained with anti-TH marmosets seemed consistent with propagation through antibody indicated an apparent decrease of TH-positive neuronal networks (Brainstem anatomy WIKI, http:// staining in the injected hemisphere (Fig 7a). Therefore, we brainstemwiki.colorado.edu/doku.php/start). In these mar- quantitated TH-positive neurons in the right and left mosets, α-synuclein pathologies were seen from the injec- hemispheres and we also quantitated pS129-positive tion sites (caudate nucleus and putamen) to neocortex, neurons, and compared them. As shown in Fig 7d – e, substantia nigra, amygdala, globus pallidus and thalamus, pS129-positive aggregates were much more abundant which are brain nuclei or regions providing direct input to in the right hemisphere than in the left hemisphere. On striatum. As projections to the striatum, nigrostriatal input, the other hand, a significant decrease in the amount of corticostriatal input, thalamostriatal input, and inputs from TH-positive neurons was detected in the right hemi- the external segment of globus pallidus and subthalamic sphere in both marmosets, although the reduction was nucleus have been reported [4, 12–14, 20, 40, 53, 59]. more prominent in the marmoset that developed more Therefore, pathological α-synuclein seemed to have spread pS129-positive aggregates following injection into both to the second connection regions from the direct input caudate nucleus and putamen. Shimozawa et al. Acta Neuropathologica Communications (2017) 5:12 Page 6 of 14 Fig. 3 Immunostainings of 14H brain sections with anti-pS129 antibody. PS129-positive inclusions observed in various brain regions. Cd: caudate nucleus, Pu: putamen, Acb: accumbens, ST: bed nucleus of the stria terminal, SNC: substantia nigra compacta, SNR: substantia nigra pars reticulata, Amy: amygdala, Thal: thalamus, Cing cx: cingulate cortex, Ins cx: insular cortex, Ent cx: entorhinal cortex, Temp cx: temporal cortex, DR: dorsal raphe nuclei, LC: locus ceruleus, CA1: hippocampal CA1, EGP: external segment of globus pallidus. Similar pS129-positive inclusions were also observed in14I marmoset brain. Scale bars: 50 μm Most of the aggregates are strongly positive for both FSB thioflavin-S and FSB (Fig 8), indicating that the inclu- and thioflavin-S sions formed in marmosets at only 3 months after in- To further investigate whether the pS129-positive ag- jection have cross-β structures similar to those found gregates in these marmosets are amyloid-like struc- in brains of patients. These results suggest that propa- tures, we tried to stain them with thioflavin S and FSB. gation and spreading of these α-synuclein pathologies Remarkably, most of these aggregates, including LB-like in vivo may be monitored by the use of amyloid- and LN-like structures, were strongly positive for both imaging probes. Fig. 4 Immunostainings of 14H brain sections with anti-α-synuclein antibodies (LB509-positive inclusions in substantia nigra and stainings with 75–91 and #2642 in caudate nucleus). Similar inclusions were observed in 14I marmoset brain. Scale bars: 50 μm Shimozawa et al. Acta Neuropathologica Communications (2017) 5:12 Page 7 of 14 Fig. 5 Immunostainings of 14H brain sections (substantia nigra) with anti-p62 and anti-ubiquitin (anti-Ub) antibodies. Diaminobenzidine staining (upper panel) and double immunofluorescence stainings (middle and lower panel) indicated colocalization of pS129-positive inclusions and p62 or ubiquitin. Similar results were obtained in other brain regions and also in 14I marmoset brain. Scale bars: 50 μm Degenerating neurons with α-synuclein inclusions may be labeling with anti-pS129 and anti-Iba1 antibodies (data not cleared by microglial cells shown). All of the inclusions seemed to be neuronal aggregates, and no astrocytic or oligodendrocytic glial inclusions Discussion were observed. In fact, most of the pS129-positive inclu- Emerging evidence indicates that intracellular amyloid-like sions were closely associated with NeuN-positive nuclei proteins have prion-like properties and propagate from cell in neuronal cells, but no such colocalization was ob- to cellbyconvertingnormalproteinsinto abnormalforms served with astrocytic marker GFAP or oligodendrocytic [18, 22, 27, 42]. This prion-like propagation may account marker CNPase (Fig 9a, b and c). Very interestingly, for the characteristic spreading of pathological proteins double labeling with LB509 and Iba1 revealed that some including α-synuclein, tau and TDP-43, and also for of the LB509-positive inclusions were colocalized with disease progression in major neurodegenerative diseases Iba1-positive microglial cells (Fig 9d, e), strongly sug- with these protein pathologies, such as Parkinson’sdisease, gesting that the inclusions or degenerating neurons with Alzheimer’s disease and amyotrophic lateral sclerosis. aggregates may be phagocytosed by microglial cells. The In this study, we tested whether inoculation of synthetic LB509-positive α-synuclein inclusions should be composed mouse α-synuclein fibrils can induce PD-like α-synuclein of endogenous marmoset α-synuclein, but not injected pathologies and prion-like propagation in two adult mouse α-synuclein, since LB509 recognizes human and marmosetsbyinjecting thefibrils into striatum (one primate α-synuclein. The colocalization was confirmed by animal was injected into caudate nucleus and the other, 3D imaging (Fig 9e). Similar colocalization of α-synuclein into caudate nucleus and putamen). Within only 3 months inclusions and microglial cells was observed by double after injection, we observed abundant phospho-α-synuclein Shimozawa et al. Acta Neuropathologica Communications (2017) 5:12 Page 8 of 14 Fig. 6 Distribution of pS129-positive α-synuclein pathologies at 3 months after mouse α-synuclein fibril injection in the two marmosets (14H and 14I). The pS129-positive phase contrast images of four coronal brain sections (a – d) acquired by using BZ-X710 microscope system are shown in red with marmoset brain sections of corresponding brain regions. Coronal brain atlas (Hardman and Ashwell, 2012) locations of interaural +12.35 mm (a), +10.80 mm (b), +07.70 mm (c) and +05.60 mm (d) are shown. Asterisks in b indicate the injection sites in caudate nucleus and/or putamen. Arrows in d indicate substantia nigra pathologies in various brain regions of both marmosets, in- type marmoset triggered PD-like α-synuclein patholo- dicating that prion-like conversion readily occurred in the gies, which propagated retrogradely to substantia nigra primate brains even within this short time-scale. Luk et al. and other input regions, and induced degeneration of and we have established a propagation model in wild-type dopaminergic neurons. Furthermore, most of the inclu- mouse [30, 33, 34, 57], but others have found it difficult to sions were positive for amyloid-sensitive dyes, such as detect the pathologies in wild-type mouse [46]. It has also thioflavin-S and FSB. This simple experiment has provided been reported that intranigral or intrastriatal inoculations direct evidence for prion-like propagation of pathological of PD-derived LB extracts in monkey resulted in pro- α-synuclein in brains of primates, and the model should gressive nigrostriatal neurodegeneration, but clearly de- be very useful for establishing in vivo imaging method- fined LB-type inclusions were not observed [44]. The ology for abnormal α-synuclein propagation and for de- results of the present study clearly demonstrate that in- velopment and evaluation of disease-modifying drugs oculation of fibrillar α-synuclein in striatum of wild- for α-synucleinopathies. Shimozawa et al. Acta Neuropathologica Communications (2017) 5:12 Page 9 of 14 Fig. 7 (See legend on next page.) Shimozawa et al. Acta Neuropathologica Communications (2017) 5:12 Page 10 of 14 (See figure on previous page.) Fig. 7 Presence of pS129-positive inclusions in TH-positive neurons and significant reduction of TH-positive neurons in the ipsilateral side of the marmosets (14H and 14I). a, Immunohistochemical staining of substantia nigra with anti-TH antibody and diaminobenzidine staining in 14H. b, Double-labeling of substantia nigra with anti-TH (green) and anti-pS129 (red) antibodies in 14H. c, High magnification of the double-labeling of substantia nigra on the ipsilateral side (indicated by the squares in b). An apparent reduction of TH-positive dopamine neurons was de- tected in the ipsilateral side of the brain compared to the contralateral side. Areas of pS129-positive inclusions and areas of TH-positive neu- rons were quantified (d – g). d, e Quantification of pS129-positive inclusions and TH-positive cells in the right and left hemispheres of 14H brain. f, g Quantifications of pS129-positive inclusions and TH-positive cells in the right and left hemispheres of the 14I. To measure positive cells, 7– 9 sections (the numbers are indicated in the columns) were selected. Larger amounts of pS129-positive inclusions were detected and significant reductions of TH-positive neurons were detected in the ipsilateral sides of substantia nigra of the two marmosets. Data were analyzed by Student's t-test. All error bars indicate means ± S.E.M. **p < 0.002, * p < 0.05. Scale bars: 50 μm In this study, we did not perform behavioral tests, but mouse models, Tg-mice overexpressing human A53T we did not observe any apparent symptoms or behavior mutant α-synuclein (such as M83 line) are considered a deficits in these marmosets, suggesting that they may good host animal for inoculation experiments, because not develop strong phenotypes within 3 months after in- disease symptoms and α-synuclein pathologies appear at oculation. It is reasonable to speculate that motor defi- about ~100 days after inoculation [43]. When Tg-marmoset cits would only be detected after the loss of more than models overexpressing human α-synuclein are available, 50% of dopamine neurons, as is the case in PD patients. it will be interesting to inject synthetic α-synuclein fi- We observed 20 – 40% decrease of TH-positive cells in brils or brain extracts from patients into these animals the right hemisphere in the animals in this study. Further to see whether the appearance of PD-like symptoms or studies will be needed to establish the relationship be- pathologies is accelerated. tween pathologies and symptoms in wild-type marmosets. By double immunolabeling of marmoset brain sections The present model should be useful for research on with LB509 and Iba1, we demonstrated that some of the PD and α-synucleinopathies, because this is a primate α-synuclein inclusions are colocalized with Iba1-positive and non-transgenic wild-type animal model, which would microglial cells. This finding suggests that inclusions or not suffer from various artifacts associated with overex- degenerating neurons with aggregates may be phagocy- pression of proteins in transgenic animals [47] or the use tosed by microglial cells. Although it has been debated of viral vector-mediated gene transfer systems. Among whether inflammation constitutes a cause or consequence Fig. 8 Fluorescence labeling of α-synuclein inclusions with β-sheet ligands in 14H (caudate nucleus). a Double staining with 0.001% thioflavin-S (left) and anti-pS129 antibody (middle). Top right and bottom right panels depict high-power photomicrographs of areas indicated by squares in the left and middle panels, respectively. A large proportion of α-synuclein inclusions stained with pS129 was labeled with thioflavin-S. b Double staining with 0.001% FSB (left) and pS129 (middle). Top right and bottom right panels depict high-power photomicrographs of areas indicated by the squares in the left and middle panels, respectively. A large proportion of α-synuclein inclusions stained with pS129 was strongly labeled with FSB. Similar labelings were observed in 14I marmoset brain. Scale bars: 50 μm Shimozawa et al. Acta Neuropathologica Communications (2017) 5:12 Page 11 of 14 Fig. 9 Double immunolabeling of α-synuclein inclusions with pS129 antibody and anti-NeuN, anti-GFAP, anti-CNPase or anti-Iba1 antibodies in 14H (caudate nucleus) a Double staining with anti-pS129 and anti-NeuN antibodies. b Double staining with anti-pS129 and anti-GFAP antibodies. c Double staining with pS129 and anti-CNPase antibodies. d Double staining with LB509 and anti-Iba1 antibodies. e, A high-power photomicrographs of the double staining with LB509 and anti-Iba1 antibodies. Similar stainings were observed in various other brain regions and also in 14I marmoset brain. Scale bars: 50 μm Shimozawa et al. Acta Neuropathologica Communications (2017) 5:12 Page 12 of 14 of PD, increasing evidence suggests that microglial cells Author details Department of Dementia and Higher Brain Function, Tokyo Metropolitan and inflammatory pathways are involved in the pathogen- Institute of Medical Science, Tokyo 156-8506, Japan. Department of esis and progression of PD [8, 9]. Indeed, activated micro- Biological Science, Tokyo Metropolitan University, Tokyo 192-0397, Japan. glia are prevalent in the most pathologically affected areas National Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science and Technology, Chiba 263-8555, Japan. in the brains of PD patients [9, 35]. Recent studies also Department of Molecular Neuroimaging, Tohoku University Graduate School demonstrated that toll-like receptor 2 may contribute to 5 of Medicine, Sendai 980-8575, Japan. Department of Pharmacology, Tohoku α-synuclein pathology in PD [10]. However, there has University Graduate School of Medicine, Sendai 980-8575, Japan. Animal Research Division, Tokyo Metropolitan Institute of Medical Science, Tokyo been no direct evidence that microglial cells are involved 156-8506, Japan. in the clearance of α-synuclein aggregates, and our findings here represent the evidence that α-synuclein Received: 18 December 2016 Accepted: 18 January 2017 aggregates or cells with inclusions are phagocytosed by microglial cells for clearance. It seems plausible that References such microglial phagocytosis of α-synuclein inclusions 1. Appel-Cresswell S, Vilarino-Guell C, Encarnacion M, Sherman H, Yu I, Shah B, may be a protective event to clear degenerating neurons Weir D, Thompson C, Szu-Tu C, Trinh J et al (2013) Alpha-synuclein p.H50Q, and reduce inflammation in the brain, but further studies a novel pathogenic mutation for Parkinson's disease. Mov Disord 28:811–813. doi:10.1002/mds.25421 will be needed to confirm this. Our marmoset model 2. 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Propagation of pathological α-synuclein in marmoset brain

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Abstract

α-Synuclein is a defining, key component of Lewy bodies and Lewy neurites in Parkinson’s disease (PD) and dementia with Lewy bodies (DLB), as well as glial cytoplasmic inclusions in multiple system atrophy (MSA). The distribution and spreading of these pathologies are closely correlated with disease progression. Recent studies have revealed that intracerebral injection of synthetic α-synuclein fibrils or pathological α-synuclein prepared from DLB or MSA brains into wild-type or transgenic animal brains induced prion-like propagation of phosphorylated α-synuclein pathology. The common marmoset is a very small primate that is expected to be a useful model of human diseases. Here, we show that intracerebral injection of synthetic α-synuclein fibrils into adult wild-type marmoset brains (caudate nucleus and/or putamen) resulted in spreading of abundant α-synuclein pathologies, which were positive for various antibodies to α-synuclein, including phospho Ser129-specific antibody, anti-ubiquitin and anti-p62 antibodies, at three months after injection. Remarkably, robust Lewy body-like inclusions were formed in tyrosine hydroxylase (TH)-positive neurons in these marmosets, strongly suggesting the retrograde spreading of abnormal α-synuclein from striatum to substantia nigra. Moreover, a significant decrease in the numbers of TH-positive neurons was observed in the injection-side of the brain, where α-synuclein inclusions were deposited. Furthermore, most of the α-synuclein inclusions were positive for 1-fluoro-2,5-bis (3-carboxy-4-hydroxystyryl) benzene (FSB) and thioflavin-S, which are dyes widely used to visualize the presence of amyloid. Thus, injection of synthetic α-synuclein fibrils into brains of non-transgenic primates induced PD-like α-synuclein pathologies within only 3 months after injection. Finally, we provide evidence indicating that neurons with abnormal α-synuclein inclusions may be cleared by microglial cells. This is the first marmoset model for α-synuclein propagation. It should be helpful in studies to elucidate mechanisms of disease progression and in development and evaluation of disease-modifying drugs for α-synucleinopathies. Keywords: α-synuclein, Parkinson, Prion, Marmoset, Circuits Introduction component of glial cytoplasmic inclusions (GCIs) in mul- Parkinson’s disease (PD) is the second most common tiple system atrophy (MSA) [54, 58]. These diseases are neurodegenerative disease after Alzheimer’s disease, and collectively referred to as α-synucleinopathies. To date, six Lewy bodies (LBs) and Lewy neurites (LNs) are character- missense mutations in the SNCA gene and occurrence of istic features of PD. Dementia with Lewy bodies (DLB) is gene multiplication have been identified in familial forms also a progressive neurodegenerative disease characterized of PD and DLB [1, 5, 24, 28, 29, 41, 52, 62]. α-Synuclein is by the appearance of LBs and LNs in cortex [17, 22]. The a small protein of 140 amino acids, which is localized in discovery of disease-associated mutation in the α-synuclein presynaptic termini, and is involved in maintenance of gene SNCA and subsequent immunostaining studies with synapses and synaptic plasticity. In PD, DLB, or MSA pa- antibodies demonstrated that α-synuclein is the major tients, it is deposited in the brain as a filamentous form component of LBs and LNs [2, 55, 56]. It is also the major with cross-β structure [51], which is abnormally phos- phorylated at Ser129 and partially ubiquitinated [15, 21]. α-Synuclein is natively unfolded, but readily assembles * Correspondence: hasegawa-ms@igakuken.or.jp into amyloid-like fibrils under appropriate conditions. Department of Dementia and Higher Brain Function, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506, Japan Pathogenic mutations affect fibril formation in vitro, either Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Shimozawa et al. Acta Neuropathologica Communications (2017) 5:12 Page 2 of 14 accelerating fibril formation [6, 7, 16] or resulting in were originally one structure and that they became sepa- formation of fibrils that are more fragile and easier to rated by the internal capsule during evolution [25]. propagate than wild-type (WT) fibrils [61]. Moreover, Thus, the marmoset has advantageous characteristics as the spreading of pathological α-synuclein is closely corre- an experimental animal to study brain networks, func- lated with disease progression; indeed, the distribution tions and disease conditions. pattern and spread of the pathologies are useful for disease Here, we investigated whether intracerebral injection of staging of sporadic PD [3, 48]. These results suggest that α-synuclein fibrils can induce PD/DLB-like pathologies in intracellular amyloid-like α-synuclein fibrils can cause PD marmoset, and we present the first marmoset model of and DLB, and spreading of α-synuclein pathology in the α-synuclein propagation. We found that marmosets devel- brain is considered to be the underlying mechanism of oped abundant phosphorylated α-synuclein pathologies, progression of these diseases. Recently, it was experi- similar to those observed in PD/DLB, in various brain mentally demonstrated that intracerebral injection of regions, including striatum, cortex and substantia nigra, at synthetic α-synuclein fibrils and/or insoluble α-synuclein only three months after injection. Remarkably, many LB- from diseased brain converts normal α-synuclein into an like inclusions are observed in tyrosine hydroxylase (TH)- abnormal form, and the abnormal α-synuclein propagates positive dopamine neurons, and a significant decrease in throughout the brain in a prion-like manner in WT TH-staining was seen in the injection hemisphere. The mouse [30, 33, 34, 57], α-synuclein transgenic mouse inclusions were also positive for fluorescent β-sheet ligands, [31, 36, 60] and monkey [44]. thioflavin-S and FSB, implying that α-synuclein deposits in Common marmoset (Callithrix jacchus) is a very small these animals should be detectable in vivo by positron new world primate, about 25 – 35 cm in height and emission tomography (PET) with a suitable small- 300 – 500 g in weight, and is far more experimentally molecular agent. Taking account of the advantages of mar- tractable than macaque monkey. Since it has high fecund- mosets over mice, we believe the current experimental ity, with a short sexual maturation period of 18 months, it model would be particularly useful to examine the relation- is attracting increasing attention as an experimental model ships between PET-detectable α-synuclein lesions and dis- of primates. In fact, a national project called Brain/ ruptions of neural networks in the absence and presence of MINDS (Brain Mapping by Integrated Neurotechnologies candidate α-synucleinopathy-modifying therapeutics. for Disease Studies) was started in 2014 in Japan to de- velop the common marmoset as a model animal for Materials and methods neuroscience [19, 38, 39]. The marmoset cortex is rela- Preparation of recombinant α-synuclein and fibrils tively smooth, but the gyrencephalic and cortical sheet is Recombinant human and mouse wild-type α-synuclein divided into functionally distinct cortical areas, as in Old and fibrils were prepared as described previously [33, 57]. World monkeys [45], and thus is suitable for studies of Briefly, purified α-synuclein (7 – 10 mg/ml) was incubated higher cognitive functions and social communication [11]. at 37 °C in a shaking incubator at 200 rpm in 30 mM Therefore, marmosets are considered to be a good experi- Tris–HCl, pH 7.5, containing 0.1% NaN , for 72 h. α- mental model animal to understand the evolution of brain Synuclein fibrils were pelleted by spinning the assembly development and function. Moreover, transgenic marmo- mixtures at 113,000 xg for 20 min, resuspended in 30 mM sets have already been generated, demonstrating the feasi- Tris–HCl buffer (pH 7.5), and sonicated for 3 min (Biomic bility of gene manipulation in this species [49]. 7040 Ultrasonic Processor, Seiko). The protein concentra- To date, mouse models have been used to investigate tions were determined by HPLC. Samples were run on brain development, circuits, and higher cognitive func- gradient 12% polyacrylamide gels and stained with Coo- tions, but they have limitations for exploration of the massie Brilliant Blue (CBB), or electrophoretically trans- evolution and development of the primate neocortex. In ferred to PVDF membranes. For immunoblotting, situ hybridization analysis of marmoset brain revealed membranes were incubated with 3% gelatin (Wako) for that the expression patterns of the genes that regulate 10 min at 37 °C, followed by overnight incubation at room brain development (such as EphA6) are different, especially temperature with primary antibodies. Next, the mem- in brain areas that have connections to the prefrontal branes were incubated for 1 hr at room temperature with cortex and are presumably involved in higher cognitive biotinylated anti-rabbit or mouse IgG (Vector Lab), then functions, although similar broad regional patterns of incubated for 30 min with avidin-horseradish peroxidase expression were observed in both species [32]. (Vector Lab), and the reaction product was visualized by A particular difference in brain development and using 0.1% 3,3-diaminobenzidine (DAB) and 0.2 mg/ml structure between mouse and marmoset is that striatum NiCl as the chromogen. For electron microscopy, sam- of marmoset is separated into caudate nucleus and puta- ples were placed on collodion-coated 300-mesh copper men, while these are not distinguishable in rodents. It grids, stained with 2% (v/v) phosphotungstate, and exam- has been considered that caudate nucleus and putamen ined with a JEOL 1200EX electron microscope. Shimozawa et al. Acta Neuropathologica Communications (2017) 5:12 Page 3 of 14 Marmosets detection and characterization of α-synuclein pathologies According to animal protection considerations based on in marmoset brains. Anti-p62 (Progen), anti-Ub (Dako, the 3R (reduce, reuse, recycle) principle, we designed the Millipore), anti-TH (Millipore), anti-NeuN (Millipore), experiment very carefully to minimize the number of anti-GFAP (Sigma), anti-CNPase (Abcam) and anti-Iba1 animals used. Two female 26-month-old marmosets (Wako) antibodies were also used. (individual recognition No. 14H and 14I; born on 4th April, 2014 and bred at the Animal Research Division, Tokyo Metropolitan Institute of Medical Science) were Immunohistochemistry used for this experiment. Marmosets were deeply anesthetized with pentobarbital injection and killed, and the brain was perfused with Stereotaxic surgery 0.1 M phosphate buffer, followed by 10% formalin neutral The marmosets were anesthetized with Ketamine Hydro- buffer solution. After fixation, whole brains were sectioned chloride (20–40 mg/kg i.m.) and Xylazine (0.05 mg/kg coronally at 50 μm using a vibratome (Leica, Wetzlar, i.m.), and Butorphanol (0.05–0.1 mg/kg i.m.). Then, 50 μL Germany). For high-sensitivity detection, free-floating aliquots of 4 mg/mL mouse α-synuclein fibrils were brain sections were treated with formic acid for 20 min, injected into both caudate nucleus (interaural +9.5 mm, washed, and boiled at 100 °C for 20 min as described [33]. Lateral 3 mm, Depth 6 mm) and putamen (interaural Sections were then incubated with 0.5% H O in methanol 2 2 +9.5 mm, Lateral 6 mm, Depth 3 mm) in the right hemi- for 30 min to inactivate endogenous peroxidases, blocked sphere of 14H brain (total 400 μg). A 50 μL aliquot was with 10% calf serum in PBS for 20 min, and incubated injected into caudate nucleus (interaural +9.5 mm, Lateral overnight with appropriate antibodies. After incubation 3 mm, Depth 6 mm) in the right hemisphere of 14I brain with the biotinylated secondary antibody for 2 h, labeling (total 200 μg). The marmosets were bred for 3 months was detected using the ABC staining kit (Vector) with after injection in a biological safety level 2 (BSL-2) envir- DAB. Sections were counterstained with hematoxylin. onment. All experimental protocols were approved by the Slides were coverslipped with mounting medium. Images Animal Care and Use Committee of Tokyo Metropolitan were observed with an all-in-one microscope/digital Institute of Medical Science (No. 16038). camera (BZ-X710; Keyence). For double-label immunofluorescence detection, brain Antibodies sections were pretreated as described above and incubated Primary antibodies used in this study are listed in Table 1. overnight at 4 °C with a cocktail of appropriate primary An anti-phosphorylated α-synuclein rabbit monoclonal antibodies. The sections were washed and incubated with antibody to pS129 (Abcam) and other anti-α-synuclein a cocktail of Alexa568-conjugated goat anti-mouse or antibodies, including LB509 [26] (a gift from Dr anti-rabbit IgG and Alexa488-conjugated goat anti-mouse Iwatsubo), 75–91 (Cosmo bio), 131–140 (Cosmo bio) or anti-rabbit or anti-guinea pig IgG (Molecular Probes). and #2642 (Cell Signaling Technology) were used for After further washing, the sections were coverslipped with Table 1 Antibodies used in this study Primary antibodies Type Source Dilution pS129 (phosphorylated a-syn) rabbit mono Abcam (ab51253) 1:2000 LB509 (human a-syn) mouse mono Gift from Dr Iwatsubo 1:1000 75–91 (a-syn 75–91) rabbit poly Cosmo bio (CAC-TIPSNP08) 1:1000 131–141 (a-syn 131–140) rabbit poly Cosmo bio (CAC-TIPSNP09) 1:1000 #2642 (a-syn) rabbit poly Cell Signaling Tech (#2642) 1:1000 Anti-p62 guinea pig poly Progen (GP62-C) 1:1000 Anti-Ub rabbit poly Dako (Z0458) 1:1000 Anti-Ub mouse mono Millipore (MAB1510) 1:1000 Anti-TH rabbit poly Millipore (AB152) 1:1000 Anti-TH mouse mono Millipore (MAB318) 1:1000 Anti-NeuN mouse mono Millipore (MAB377) 1:1000 Anti-GFAP mouse mono Sigma (G3893) 1:1000 Anti-CNPase mouse mono Abcam (ab6319) 1:200 Anti-Iba1 rabbit poly Wako (016–20001) 1:1000 Shimozawa et al. Acta Neuropathologica Communications (2017) 5:12 Page 4 of 14 non-fluorescent mounting media (VECTASHIELD; mouse α-synuclein fibrils instead of human α-synuclein Vector Laboratories) and observed with the BZ-X710. fibrils for injection into marmoset brains in this experi- To measure positive cells, 7–9 sections of substantia ment. This had the advantage that we could investigate nigra were randomly selected, and all images were cap- whether endogenous marmoset α-synuclein is deposited tured with BZ-X710 microscope using the same settings. in brains injected with mouse α-synuclein fibrils, be- The areas of pS129-positive cells and TH-positive cells cause the two can be distinguished with antibodies such in the right and left substantia nigra were extracted and as LB509. Recombinant mouse α-synuclein and the fibrils quantified by BZ-H3C Hybrid Cell Count Software were prepared as described, and images of purified recom- (Keyence). binant mouse α-synuclein and the fibrils are shown in Fig 2. EM pictures of the injected sample showed that Thioflavin-S and FSB stainings most of the fibrils were straight, 5 – 10 nm in width, and Thioflavin-S and FSB [23, 50] were purchased from 50 – 300 nm in length (Fig 2C). The prion-like seeding Sigma-Algrich and Dojindo, respectively. For fluorescence activity of the fibrils to convert normal α-synuclein into labeling with β-sheet ligands, thioflavin-S and FSB, brain abnormal form was checked in our cultured cell model sections were mounted on a glass slide and dried with [37] and mouse model [57] (data not shown). We injected warm air. Sections were incubated in 20% ethanol con- 200– 400 μgof the α-synuclein fibrils; this amount was taining 0.001% β-sheet ligands at room temperature for chosen based on the amount used in the previous mouse 30 min. The samples were rinsed with 20% ethanol for experiments (10 μg fibrils/0.3 g mouse brain vs 200 μg 5 min, dipped into distilled water twice for 3 min each, fibrils/7 g marmoset brain). and mounted in VECTASHIELD. Fluorescence images were captured using a BZ-X710 fluorescence microscope PD-like pathologies in marmoset brain within only equipped with Filter set ET-ECFP (Chroma Technology) 3 months after injection for thioflavin-S and FSB. After fluorescence microscopy, Three months after injection, formalin-fixed marmoset all sections labeled with ligands were autoclaved for anti- brain sections were prepared and immunostained with gen retrieval, immunostained with the pS129 antibody, anti-phospho-Ser129 of α-synuclein (pS129). Surprisingly, and examined using the BZ-X710 instrument. abundant pS129-positive structures were observed throughout the brain regions, including the injection Results sites in the caudate nucleus and putamen, accumbens, Inoculation of mouse α-synuclein fibrils into marmoset brain bed nucleus of the stria terminalis, substantia nigra, amyg- Marmoset α-synuclein, mouse α-synuclein and human dala, a wide region of cortex, thalamus, globus pallidus, α-synuclein share 96 – 97% amino acid sequence hom- dorsal raphe nuclei, raphe nucleus and hippocampal CA1 ology (Fig 1), but marmoset and mouse α-synuclein pro- in both marmosets (Fig 3). These abnormal α-synuclein teins both have a threonine residue at amino acid pathologies were also positive with other anti-α-synuclein position 53, which is an aggregation-prone mutation in antibodies (Fig 4), including LB509. The results indicated familial Parkinson’s disease [41]. Therefore, we used that endogenous marmoset α-synuclein is converted into Fig. 1 Comparison of amino acid sequences of human (Homo sapiens), marmoset (Callithrix jacchus)and mouse (Mus musculus) α-synuclein. Amino acids in human α-synuclein that differ from those of marmoset and mouse α-synuclein are indicated in red, while amino acids in marmoset and mouse α-synuclein that differ from those in human α-synuclein are indicated in green and blue, respectively. Epitopes of α-synuclein antibodies used in this study are also indicated Shimozawa et al. Acta Neuropathologica Communications (2017) 5:12 Page 5 of 14 Fig. 2 Characterization of purified mouse α-synuclein and fibrils. a, purified monomeric human α-synuclein (lane 1), mouse α-synuclein (lane 2) and mouse α-synuclein fibril (lane 3) samples were analyzed by CBB staining (0.5 μgofprotein/lane). b, results of immunoblotting with anti-α-synuclein antibodies (131–140 and LB509) (B, 0.01 μgofprotein/lane). c, electron microscopy of α-synuclein fibrils injected into marmoset brains in this study. Negatively stained short fibrils, 50 –300 nm in length, were observed. Scale bar: 100 nm an abnormal form by inoculation of mouse α-synuclein fi- regions over the 3-month period. Overall, the spreading brils and deposited in the brain. Most of the inclusions pattern of pathological α-synuclein seemed to be the were also positive for antibodies to ubiquitin and p62 same as in mouse brain [30, 33, 57], and was consistent (Fig 5), as is the case in the brains of patients. Labeling of with the spreading pattern of retrograde tracers [40]. the inclusions with these antibodies was observed without It is noteworthy that abundant LB-like round pS129- pretreatment of sections with formic acid and boiling, but positive inclusions were detected in substantia nigra of the specificity and sensitivity were both increased by the these marmosets (Fig 3, 4, 5 and 7). The nigral LB-like pretreatment (data not shown). inclusions were more prominent in the marmoset injected into both caudate nucleus and putamen than in the α-Synuclein propagated retrogradely through neuronal marmoset injected only into caudate nucleus. Double networks labeling of the inclusions with anti-tyrosine-hydroxylase The distributions of the pathologies were similar in these (TH) antibody confirmed that the inclusions are formed in two marmosets 14H and 14I, although more abundant TH-positive dopaminergic neurons (Fig 7b, c), indicating and severe pathologies were observed in the marmoset that pathological α-synuclein was propagated retrogradely 14H (Fig 6), in which α-synuclein fibrils were injected at from striatum to nigral neurons. both caudate nucleus and putamen. The α-synuclein pathologies were robust in the injected right hemisphere, but also present in the left hemisphere, although to a Degeneration of dopaminergic neurons with pS129- much lesser extent than in the right hemisphere (Fig 6). positive inclusions The spreading of α-synuclein pathology observed in the Macroscopical observation of sections stained with anti-TH marmosets seemed consistent with propagation through antibody indicated an apparent decrease of TH-positive neuronal networks (Brainstem anatomy WIKI, http:// staining in the injected hemisphere (Fig 7a). Therefore, we brainstemwiki.colorado.edu/doku.php/start). In these mar- quantitated TH-positive neurons in the right and left mosets, α-synuclein pathologies were seen from the injec- hemispheres and we also quantitated pS129-positive tion sites (caudate nucleus and putamen) to neocortex, neurons, and compared them. As shown in Fig 7d – e, substantia nigra, amygdala, globus pallidus and thalamus, pS129-positive aggregates were much more abundant which are brain nuclei or regions providing direct input to in the right hemisphere than in the left hemisphere. On striatum. As projections to the striatum, nigrostriatal input, the other hand, a significant decrease in the amount of corticostriatal input, thalamostriatal input, and inputs from TH-positive neurons was detected in the right hemi- the external segment of globus pallidus and subthalamic sphere in both marmosets, although the reduction was nucleus have been reported [4, 12–14, 20, 40, 53, 59]. more prominent in the marmoset that developed more Therefore, pathological α-synuclein seemed to have spread pS129-positive aggregates following injection into both to the second connection regions from the direct input caudate nucleus and putamen. Shimozawa et al. Acta Neuropathologica Communications (2017) 5:12 Page 6 of 14 Fig. 3 Immunostainings of 14H brain sections with anti-pS129 antibody. PS129-positive inclusions observed in various brain regions. Cd: caudate nucleus, Pu: putamen, Acb: accumbens, ST: bed nucleus of the stria terminal, SNC: substantia nigra compacta, SNR: substantia nigra pars reticulata, Amy: amygdala, Thal: thalamus, Cing cx: cingulate cortex, Ins cx: insular cortex, Ent cx: entorhinal cortex, Temp cx: temporal cortex, DR: dorsal raphe nuclei, LC: locus ceruleus, CA1: hippocampal CA1, EGP: external segment of globus pallidus. Similar pS129-positive inclusions were also observed in14I marmoset brain. Scale bars: 50 μm Most of the aggregates are strongly positive for both FSB thioflavin-S and FSB (Fig 8), indicating that the inclu- and thioflavin-S sions formed in marmosets at only 3 months after in- To further investigate whether the pS129-positive ag- jection have cross-β structures similar to those found gregates in these marmosets are amyloid-like struc- in brains of patients. These results suggest that propa- tures, we tried to stain them with thioflavin S and FSB. gation and spreading of these α-synuclein pathologies Remarkably, most of these aggregates, including LB-like in vivo may be monitored by the use of amyloid- and LN-like structures, were strongly positive for both imaging probes. Fig. 4 Immunostainings of 14H brain sections with anti-α-synuclein antibodies (LB509-positive inclusions in substantia nigra and stainings with 75–91 and #2642 in caudate nucleus). Similar inclusions were observed in 14I marmoset brain. Scale bars: 50 μm Shimozawa et al. Acta Neuropathologica Communications (2017) 5:12 Page 7 of 14 Fig. 5 Immunostainings of 14H brain sections (substantia nigra) with anti-p62 and anti-ubiquitin (anti-Ub) antibodies. Diaminobenzidine staining (upper panel) and double immunofluorescence stainings (middle and lower panel) indicated colocalization of pS129-positive inclusions and p62 or ubiquitin. Similar results were obtained in other brain regions and also in 14I marmoset brain. Scale bars: 50 μm Degenerating neurons with α-synuclein inclusions may be labeling with anti-pS129 and anti-Iba1 antibodies (data not cleared by microglial cells shown). All of the inclusions seemed to be neuronal aggregates, and no astrocytic or oligodendrocytic glial inclusions Discussion were observed. In fact, most of the pS129-positive inclu- Emerging evidence indicates that intracellular amyloid-like sions were closely associated with NeuN-positive nuclei proteins have prion-like properties and propagate from cell in neuronal cells, but no such colocalization was ob- to cellbyconvertingnormalproteinsinto abnormalforms served with astrocytic marker GFAP or oligodendrocytic [18, 22, 27, 42]. This prion-like propagation may account marker CNPase (Fig 9a, b and c). Very interestingly, for the characteristic spreading of pathological proteins double labeling with LB509 and Iba1 revealed that some including α-synuclein, tau and TDP-43, and also for of the LB509-positive inclusions were colocalized with disease progression in major neurodegenerative diseases Iba1-positive microglial cells (Fig 9d, e), strongly sug- with these protein pathologies, such as Parkinson’sdisease, gesting that the inclusions or degenerating neurons with Alzheimer’s disease and amyotrophic lateral sclerosis. aggregates may be phagocytosed by microglial cells. The In this study, we tested whether inoculation of synthetic LB509-positive α-synuclein inclusions should be composed mouse α-synuclein fibrils can induce PD-like α-synuclein of endogenous marmoset α-synuclein, but not injected pathologies and prion-like propagation in two adult mouse α-synuclein, since LB509 recognizes human and marmosetsbyinjecting thefibrils into striatum (one primate α-synuclein. The colocalization was confirmed by animal was injected into caudate nucleus and the other, 3D imaging (Fig 9e). Similar colocalization of α-synuclein into caudate nucleus and putamen). Within only 3 months inclusions and microglial cells was observed by double after injection, we observed abundant phospho-α-synuclein Shimozawa et al. Acta Neuropathologica Communications (2017) 5:12 Page 8 of 14 Fig. 6 Distribution of pS129-positive α-synuclein pathologies at 3 months after mouse α-synuclein fibril injection in the two marmosets (14H and 14I). The pS129-positive phase contrast images of four coronal brain sections (a – d) acquired by using BZ-X710 microscope system are shown in red with marmoset brain sections of corresponding brain regions. Coronal brain atlas (Hardman and Ashwell, 2012) locations of interaural +12.35 mm (a), +10.80 mm (b), +07.70 mm (c) and +05.60 mm (d) are shown. Asterisks in b indicate the injection sites in caudate nucleus and/or putamen. Arrows in d indicate substantia nigra pathologies in various brain regions of both marmosets, in- type marmoset triggered PD-like α-synuclein patholo- dicating that prion-like conversion readily occurred in the gies, which propagated retrogradely to substantia nigra primate brains even within this short time-scale. Luk et al. and other input regions, and induced degeneration of and we have established a propagation model in wild-type dopaminergic neurons. Furthermore, most of the inclu- mouse [30, 33, 34, 57], but others have found it difficult to sions were positive for amyloid-sensitive dyes, such as detect the pathologies in wild-type mouse [46]. It has also thioflavin-S and FSB. This simple experiment has provided been reported that intranigral or intrastriatal inoculations direct evidence for prion-like propagation of pathological of PD-derived LB extracts in monkey resulted in pro- α-synuclein in brains of primates, and the model should gressive nigrostriatal neurodegeneration, but clearly de- be very useful for establishing in vivo imaging method- fined LB-type inclusions were not observed [44]. The ology for abnormal α-synuclein propagation and for de- results of the present study clearly demonstrate that in- velopment and evaluation of disease-modifying drugs oculation of fibrillar α-synuclein in striatum of wild- for α-synucleinopathies. Shimozawa et al. Acta Neuropathologica Communications (2017) 5:12 Page 9 of 14 Fig. 7 (See legend on next page.) Shimozawa et al. Acta Neuropathologica Communications (2017) 5:12 Page 10 of 14 (See figure on previous page.) Fig. 7 Presence of pS129-positive inclusions in TH-positive neurons and significant reduction of TH-positive neurons in the ipsilateral side of the marmosets (14H and 14I). a, Immunohistochemical staining of substantia nigra with anti-TH antibody and diaminobenzidine staining in 14H. b, Double-labeling of substantia nigra with anti-TH (green) and anti-pS129 (red) antibodies in 14H. c, High magnification of the double-labeling of substantia nigra on the ipsilateral side (indicated by the squares in b). An apparent reduction of TH-positive dopamine neurons was de- tected in the ipsilateral side of the brain compared to the contralateral side. Areas of pS129-positive inclusions and areas of TH-positive neu- rons were quantified (d – g). d, e Quantification of pS129-positive inclusions and TH-positive cells in the right and left hemispheres of 14H brain. f, g Quantifications of pS129-positive inclusions and TH-positive cells in the right and left hemispheres of the 14I. To measure positive cells, 7– 9 sections (the numbers are indicated in the columns) were selected. Larger amounts of pS129-positive inclusions were detected and significant reductions of TH-positive neurons were detected in the ipsilateral sides of substantia nigra of the two marmosets. Data were analyzed by Student's t-test. All error bars indicate means ± S.E.M. **p < 0.002, * p < 0.05. Scale bars: 50 μm In this study, we did not perform behavioral tests, but mouse models, Tg-mice overexpressing human A53T we did not observe any apparent symptoms or behavior mutant α-synuclein (such as M83 line) are considered a deficits in these marmosets, suggesting that they may good host animal for inoculation experiments, because not develop strong phenotypes within 3 months after in- disease symptoms and α-synuclein pathologies appear at oculation. It is reasonable to speculate that motor defi- about ~100 days after inoculation [43]. When Tg-marmoset cits would only be detected after the loss of more than models overexpressing human α-synuclein are available, 50% of dopamine neurons, as is the case in PD patients. it will be interesting to inject synthetic α-synuclein fi- We observed 20 – 40% decrease of TH-positive cells in brils or brain extracts from patients into these animals the right hemisphere in the animals in this study. Further to see whether the appearance of PD-like symptoms or studies will be needed to establish the relationship be- pathologies is accelerated. tween pathologies and symptoms in wild-type marmosets. By double immunolabeling of marmoset brain sections The present model should be useful for research on with LB509 and Iba1, we demonstrated that some of the PD and α-synucleinopathies, because this is a primate α-synuclein inclusions are colocalized with Iba1-positive and non-transgenic wild-type animal model, which would microglial cells. This finding suggests that inclusions or not suffer from various artifacts associated with overex- degenerating neurons with aggregates may be phagocy- pression of proteins in transgenic animals [47] or the use tosed by microglial cells. Although it has been debated of viral vector-mediated gene transfer systems. Among whether inflammation constitutes a cause or consequence Fig. 8 Fluorescence labeling of α-synuclein inclusions with β-sheet ligands in 14H (caudate nucleus). a Double staining with 0.001% thioflavin-S (left) and anti-pS129 antibody (middle). Top right and bottom right panels depict high-power photomicrographs of areas indicated by squares in the left and middle panels, respectively. A large proportion of α-synuclein inclusions stained with pS129 was labeled with thioflavin-S. b Double staining with 0.001% FSB (left) and pS129 (middle). Top right and bottom right panels depict high-power photomicrographs of areas indicated by the squares in the left and middle panels, respectively. A large proportion of α-synuclein inclusions stained with pS129 was strongly labeled with FSB. Similar labelings were observed in 14I marmoset brain. Scale bars: 50 μm Shimozawa et al. Acta Neuropathologica Communications (2017) 5:12 Page 11 of 14 Fig. 9 Double immunolabeling of α-synuclein inclusions with pS129 antibody and anti-NeuN, anti-GFAP, anti-CNPase or anti-Iba1 antibodies in 14H (caudate nucleus) a Double staining with anti-pS129 and anti-NeuN antibodies. b Double staining with anti-pS129 and anti-GFAP antibodies. c Double staining with pS129 and anti-CNPase antibodies. d Double staining with LB509 and anti-Iba1 antibodies. e, A high-power photomicrographs of the double staining with LB509 and anti-Iba1 antibodies. Similar stainings were observed in various other brain regions and also in 14I marmoset brain. Scale bars: 50 μm Shimozawa et al. Acta Neuropathologica Communications (2017) 5:12 Page 12 of 14 of PD, increasing evidence suggests that microglial cells Author details Department of Dementia and Higher Brain Function, Tokyo Metropolitan and inflammatory pathways are involved in the pathogen- Institute of Medical Science, Tokyo 156-8506, Japan. Department of esis and progression of PD [8, 9]. Indeed, activated micro- Biological Science, Tokyo Metropolitan University, Tokyo 192-0397, Japan. glia are prevalent in the most pathologically affected areas National Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science and Technology, Chiba 263-8555, Japan. in the brains of PD patients [9, 35]. Recent studies also Department of Molecular Neuroimaging, Tohoku University Graduate School demonstrated that toll-like receptor 2 may contribute to 5 of Medicine, Sendai 980-8575, Japan. Department of Pharmacology, Tohoku α-synuclein pathology in PD [10]. However, there has University Graduate School of Medicine, Sendai 980-8575, Japan. Animal Research Division, Tokyo Metropolitan Institute of Medical Science, Tokyo been no direct evidence that microglial cells are involved 156-8506, Japan. in the clearance of α-synuclein aggregates, and our findings here represent the evidence that α-synuclein Received: 18 December 2016 Accepted: 18 January 2017 aggregates or cells with inclusions are phagocytosed by microglial cells for clearance. It seems plausible that References such microglial phagocytosis of α-synuclein inclusions 1. Appel-Cresswell S, Vilarino-Guell C, Encarnacion M, Sherman H, Yu I, Shah B, may be a protective event to clear degenerating neurons Weir D, Thompson C, Szu-Tu C, Trinh J et al (2013) Alpha-synuclein p.H50Q, and reduce inflammation in the brain, but further studies a novel pathogenic mutation for Parkinson's disease. Mov Disord 28:811–813. doi:10.1002/mds.25421 will be needed to confirm this. Our marmoset model 2. 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