Human α-synuclein overexpression in a mouse model of Parkinson's disease leads to vascular pathology, blood brain barrier leakage and pericyte activation

Abstract

The pathological hallmark of Parkinson's disease (PD) is the formation of Lewy bodies containing aggregated alpha-synuclein (α-syn). Although PD is associated with these distinct histological changes, other pathological features such as microvascular alterations have been linked to neurodegeneration. These changes need to be investigated as they create a hostile brain microenvironment and may contribute to the development and progression of the disease. We use a human α-syn overexpression mouse model that recapitulates some of the pathological features of PD in terms of progressive aggregation of human α-syn, impaired striatal dopamine fiber density, and an age-dependent motor deficit consistent with an impaired dopamine release. We demonstrate for the first time in this model a compromised blood-brain barrier integrity and dynamic changes in vessel morphology from angiogenesis at earlier stages to vascular regression at later stages. The vascular alterations are accompanied by a pathological activation of pericytes already at an early stage without changing overall pericyte density. Our data support and further extend the occurrence of vascular pathology as an important pathophysiological aspect in PD. The model used provides a powerful tool to investigate disease-modifying factors in PD in a temporal sequence that might guide the development of new treatments.

Figure 1

Progressive accumulation and aggregation of α-syn and p-α-syn in the striatum of the TG mice. (a) Reconstructed confocal image showing the fluorescence density level of α-syn-GFP and pS129-α-syn in the dorsolateral striatum of TG mice at age 3, 8, and 13 months (Z thickness 4 μm, step size 0.3 μm). Scale bar: 20 μm. (b) Corresponding quantification of the α-syn-GFP and pS129-α-syn fluorescence density in the dorsolateral striatum of TG mice at age of 3 months (n = 3), 8 months (n = 3) and 13 months (n = 3). One-way ANOVA: *p-value < 0.05. (c) Fractionation of 1% Triton-X-100 soluble, and insoluble α-syn solubilized in 8 M urea, 5% SDS. Human α-syn-GFP and human pS129-α-syn-GFP protein was quantified in Image J from western blots based on the mobility compared to the mouse α-syn. Images from the western blots of the Triton X-100 soluble human α-syn compared to human α-syn and pS129-α-syn in the Triton X-100 insoluble fraction. The full-length blots are presented in Supplementary Fig. 1c. Data in (c) were quantified in (d), two way-ANOVA, Tukey multiple correction analysis: *p-value < 0.05. Human α-syn was quantified by western blot in the Triton X-100 soluble, and wash 4 fractions (Supplementary Fig. 1b). α-syn-GFP = alpha-synuclein green fluorescent protein, pS129-α-syn = phospho S129-alpha-synuclein.

Figure 2

TH cell survival and reduction in TH and DAT density in TG mice. (a) TH⁺ staining and TH⁺ cell count in the SNpc of TG and WT mice at age 13 months (n = 4 WT, 5 TG). (b) TH⁺ fibre optical density in the striatum of TG and WT mice at age 13 months (n = 5 WT, 5 TG). (c) DAT⁺ fibre optical density in the striatum of the TG and WT mice at age 13 months (n = 3 WT, 5 TG). Two-tailed student’s t-test: *p-value < 0.05, **p < 0.01. Scale bar: 50 μm. DAT = Dopamine transporter, TH = Tyrosine hydroxylase.

Figure 3

Progressively impaired motor function in TG mice. Open field motor test (left panel) showing distance travelled by WT and TG mice at naive condition and after amphetamine injection (5 mg/ kg) at (a) age of 3, (b) age of 8 and (c) age of 13 months, two-way ANOVA: *p-value < 0.05, **p < 0.01, ****p < 0.0001. Rotarod motor test (right panel) illustrating the latency to fall of accelerated rod in rotarod test at (a) age of 3 months (n = 3 WT, 5 TG), (b) age of 8 months (n = 7 WT, 7 TG) and (c) age of 13 months (n = 5 WT, 10 TG). Two-tailed student’s t-test: *p < 0.05. AMPH = amphetamine, m = meter, s = seconds.

Figure 4

Increased fibrinogen leakage in the dorsal striatum of TG mice. Confocal images illustrating fibrinogen leakage in WT and TG mice and respective quantification showing extravascular fibrinogen density at (a) age of 3 months (n = 3 WT, 3 TG); (b) age of 8 months (n = 4 WT, 4 TG); and (c) age of 13 months (n = 4 WT, 4 TG). Two-tailed student’s t-test: *p-value < 0.05. Scale bar: 50 μm. PDCLX = Podocalyxin.

Figure 5

Alterations in striatal vessel and pericyte density in TG mice. (a–c) Confocal images illustrating PDCLX⁺ vessel density in WT and TG mice and quantification at (a) age of 3 months (n = 6 WT, 9 TG); (b) age of 8 months (n = 4 WT, 4 TG); and (c) age of 13 months (n = 7 WT, 8 TG). (d–f) Confocal images showing CD13⁺ pericyte density in WT and TG mice and quantification (d) at age of 3 months (n = 4 WT, 6 TG); (e) at age of 8 months (n = 4 WT, 4 TG); and (f) at age of 13 months (n = 7 WT, 5 TG). Two-tailed student’s t-test: *p-value < 0.05. Scale bar: 100 μm.

Figure 6

Reduction of Col IV in TG mice at late age. (a) Confocal images illustrating Col IV density and its proportion in relation to the PDCLX⁺ vessels in WT (left panel) and TG mice (right panel). (b) Quantification of Col IV density and its proportion in relation to the PDCLX⁺ vessels in WT and TG mice at age of 3 months (n = 3 WT, 3 TG); age of 8 months (n = 3 WT, 3 TG); and age of 13 months (n = 3 WT, 3TG). Two-tailed student’s t-test: *p-value < 0.05. Scale bar: 50 μm. PDCLX = Podocalyxin, Col IV = Collagen IV.

Figure 7

Activated NG2⁺ pericytes in the striatum of TG mice. Confocal images showing NG2⁺ pericyte coverage (grey) of PDCLX⁺ vessels (red) in WT and TG mice and quantification of NG2⁺ pericyte density at (a) age of 3 months (n = 3 WT, 3 TG); (b) age of 8 months (n = 4 WT, 3 TG); and (c) age of 13 months (n = 4 WT, 4 TG). Confocal images showing NG2⁺ pericyte coverage (grey) of PDCLX⁺ vessels with α-syn-GFP in TG mice at age of 3 (d), 8 (e) and 13 (f) months. Two-tailed student’s t-test: *p-value < 0.05, ***p-value < 0.001. Scale bar: large image (a–f) 50 μm; image box (a–f) 20 μm. PDCLX = Podocalyxin, NG2 = Neuron-glial antigen 2, α-syn-GFP = alpha-synuclein green fluorescent protein.

Figure 8

α-syn-GFP and pS129-α-syn inclusions are present in endothelial cells but not in pericytes. (a) Confocal pictures illustrating that α-syn-GFP does not co-localize with PDGFRβ⁺ pericytes (b) but shows co-localization with PDCLX⁺ blood vessels in the striatum of TG mice at age of 3, 8, and 13 months. (c) Confocal images showing pS129-α-syn does no colocalizate with CD13⁺ pericytes (d) but it colocalizes with CD31⁺ vessels in the striatum of TG mice at age of 3, 8 and 13 months. PDGFRβ = Platelet-derived growth factor receptor-beta, PDCLX = Podocalyxin, α-syn-GFP = alpha-synuclein green fluorescent protein, pS129-α-syn = phospho S129-alpha-synuclein. Scale bar: 10 µm.

Figure 9

Summary of the key findings. Human α-syn overexpression mouse model displays early blood–brain barrier leakage, stage-dependent dynamic changes in vessel morphology and pathological activation of pericytes.