Intrastriatal alpha-synuclein fibrils in monkeys: spreading, imaging and neuropathological changes

Abstract

Several studies have demonstrated that intrastriatal injections of fibrillar α-synuclein in rodent brain induced a Parkinson's disease-like propagation of Lewy body pathology with significant nigrostriatal neurodegeneration. This study evaluated the pathological features when exogenous α-synuclein preformed fibrils were injected into the putamen of non-human primates. Eight cynomolgus monkeys received unilateral intraputamen injections of α-synuclein preformed fibrils and four monkeys received sham surgery. Monkeys were assessed with 123I-PE2I single-photon emission computerized tomography scans targeting the dopamine transprter at baseline, 3, 6, 9, 12, and 15 months. Imaging revealed a robust increase in dopamine transporter binding, an effect confirmed by port-mortem immunohistochemical analyses, suggesting that upregulation of dopamine transporter occurs as part of an early pathological process. Histochemistry and immunohistochemistry revealed that α-synuclein preformed fibrils injections into the putamen induced intraneuronal inclusions positive for phosphorylated α-synuclein in ipsilateral substantia nigra and adjacent to the injection site. α-Synuclein inclusions were thioflavin-S-positive suggesting that the inclusions induced by α-synuclein preformed fibrils exhibited pathological properties similar to amyloid-like Lewy body pathology in Parkinson's disease brains. The α-synuclein preformed fibrils resulted in Lewy pathology in the ipsilateral substantia nigra with significant reduction (-29.30%) of dopaminergic neurons as compared with controls. Nigral neurons with α-synuclein inclusions exhibited a phenotypic downregulation of the dopamine markers tyrosine hydroxylase and Nurr1. Taken together, our findings demonstrate that α-synuclein preformed fibrils induce a synucleinopathy in non-human primates with authentic Lewy pathology and nigrostriatal changes indicative of early Parkinson's disease.

Keywords: Lewy bodies; neurodegeneration; nigrostriatal system; propagation; α-synuclein.

Figure 1

Axial, sagittal, and coronal views of PE2I SPECT scans. There were initial decreases in binding followed by consistent increases in binding for the full 15 months (mo) of scans. v = standardized uptake value.

Figure 2

Microscopy images show DAT staining in monkeys receiving the sham surgery (control; A and B) and α-syn PFFs (C and D). The striatal DAT staining was light and homogenous in the monkey with sham surgery (A and B). Intensive DAT staining displayed in both caudate nuclei and putamen in monkeys receiving PFFs (C and D). Scale bar in D = 830 μm (applies to all). Quantitative optical density measurement (E) revealed further that the optical density of DAT staining was higher in striatum of monkeys receiving the α-syn PFFs than sham surgery (E; P < 0.01). AGSU = arbitrary greyscale units.

Figure 3

Sections from putamen show phosphorylated α-syn immunostaining in monkeys received α-syn PFFs (A–C) and sham surgery (D). The cells labelled with α-syn phosphorylated on Ser129 (p-S129-α-syn) were distributed around injected area (A) and displayed a strong dark immunostaining of α-syn (B). The p-S129-α-syn immuno-labelled fibres were detected far from injection area (C). There was an undetectable p-S129-α-syn in monkeys with sham surgery (D). Scale bar in D = 200 µm for A, 50 µm for B, and 20 µm for C.

Figure 4

Microscopy images of substantia nigra. Shown are α-syn phosphorylated on Ser129 (p-S129-α-syn) immunoreactivity (A–K and M–P) and thioflavin S staining (L) from monkeys received α-syn PFFs (B–L), sham surgery (A) and patient with Parkinson’s disease (M–P). Abundant p-S129-α-syn immunoreactive cells were distributed in substantia nigra pars compacta (B and C). These cells exhibited several morphological features: granules (arrows, D–F) with cytoplasmic staining, typical Lewy body with absent cytoplasm (G), and whole cells filled with p-S129-α-syn-immunoreactive products (H and I). Fragmental p-S129-α-syn-immunoreactive neuropil threads were observed in substantia nigra (J and K). Thioflavin S-positive inclusions were detected in substantia nigra (L). These pathological characteristics observed from monkeys received α-syn PFFs were similar to the Parkinson’s disease; p-S129-α-syn immunoreactive products displayed granules (arrow, M), whole cell shape (arrow, N), Lewy body-like (arrow, O), and fragmental fibre (arrow, P). Scale bar in K = 20 μm and applies to D–P; 100 μm for C; and 500 μm for A and B.

Figure 5

Patterns of TH immunoreactivities in substantia nigra from monkeys receiving sham surgery (control; A and B) and α-syn PFFs (C and D). There was a clear reduction of TH immunoreactivity in α-syn PFFs injected monkeys (arrows, C) compared with control monkeys (A). Some nigral neurons displayed light TH immunoreactivities with absent processes (arrows, D). Scale bar in D = 50 μm for B and 1.6 mm for A and C. (E) The number of TH-immunoreactive (-ir) neurons was reduced in the ipsilateral (PFFs, *P < 0.05) but not contralateral (P > 0.05) substantia nigra as compared with controls. Data are mean ± standard deviation.

Figure 6

Laser confocal microscopic images of substantia nigra. Images are from monkeys receiving PFFs (A–C and G–I) and sham surgery (control; D–F and J–L) illustrating tyrosine hydroxylase (TH: red; A and D), Nurr1 (red; G and J), phosphorylated α-syn (P-α-syn: green; B, E, H and K), and the co-localization (merged; C, F, I and L). Note that TH immunofluorescence intensity was severely diminished (arrows; A and C) in the neurons with p-S129-α-syn (arrows; B and C) immunoreactive inclusions but not in neurons without P-α-syn-immunoreactive inclusions in monkeys received α-syn PFFs. Similarly, Nurr1 immunofluorescence intensity was severely diminished in the neurons (arrows; G and I) with p-α-syn immunoreactive inclusions (arrows; H and I) but not in neurons without P-α-syn immunoreactive inclusions. In contrast, no P-α-syn immunoreactive inclusions were observed in monkeys with sham surgery (E and K). Scale bar in L = 110 μm (applies to all). Quantitative measurement further revealed that the optic densities of TH (M) and Nurr1 (N) were significantly decreased in the neurons with P-α-syn immunopositive (P-α-syn⁺) but not in neurons with P-α-syn immunonegative (P-α-syn^-) inclusion as compared with monkeys with sham surgery. ^***P < 0.001, compared with sham surgery. AFU = arbitrary fluorescence units.