Propagation of pathological α-synuclein in marmoset brain

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: Circuits; Marmoset; Parkinson; Prion; α-synuclein.

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

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 μg of protein/lane). b, results of immunoblotting with anti-α-synuclein antibodies (131–140 and LB509) (B, 0.01 μg of protein/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

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 in 14I marmoset brain. Scale bars: 50 μm

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

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

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

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 detected in the ipsilateral side of the brain compared to the contralateral side. Areas of pS129-positive inclusions and areas of TH-positive neurons 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

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

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