Systemic inflammation accelerates neurodegeneration in a rat model of Parkinson's disease overexpressing human alpha synuclein

Abstract

Increasing efforts have been made to elucidate how genetic and environmental factors interact in Parkinson's disease (PD). In the present study, we assessed the development of symptoms on a genetic PD rat model that overexpresses human α-synuclein (Snca^+/+) at a presymptomatic age, exposed to a pro-inflammatory insult by intraperitoneal injection of lipopolysaccharide (LPS), using immunohistology, high-dimensional flow cytometry, constant potential amperometry, and behavioral analyses. A single injection of LPS into WT and Snca^+/+ rats triggered long-lasting increase in the activation of pro-inflammatory microglial markers, monocytes, and T lymphocytes. However, only LPS Snca^+/+ rats showed dopaminergic neuronal loss in the substantia nigra pars compacta (SNpc), associated with a reduction in the release of evoked dopamine in the striatum. No significant changes were observed in the behavioral domain. We propose our double-hit animal as a reliable model to investigate the mechanisms whereby α-synuclein and inflammation interact to promote neurodegeneration in PD.

Fig. 1. Monocyte and microglial activation in CNS 3 months after LPS administration.

a Representative flow cytometry dot plots of isolated cells illustrating the gating strategy to determine profiles based on CD45^lowCD11b⁺ for microglia, CD45^highCD11b^high macrophages for the central nervous system (CNS)-associated phagocytes, and CD45^highCD11b− cells. b Percentage of CD45^highCD11b^high cells in the SNpc (left) and in the striatum (right). Error bars represent ±SEM. (n = 5 SAL WT, n = 5 LPS WT, n = 6 SAL Snca^+/+, n = 6 LPS Snca^+/+; Ordinary two-way ANOVA for Genotype vs Treatment; SNpc: Interaction, F (1, 18) = 0.003483; Genotype, F (1, 18) = 79.06, p < 0.001; Treatment, F (1, 18) = 18.48, p = 0.0004; *p < 0.05, ****p < 0.0001, with Bonferroni’s post hoc multiple comparisons test; striatum: Interaction, F (1, 18) = 0.02436; Genotype, F (1, 18) = 52.37, p < 0.0001; Treatment, F (1, 18) = 13.76, p = 0.0016; ***p < 0.001, ****p < 0.0001, with Bonferroni’s post hoc multiple comparisons test). c Percentage of CD45^lowCD11b⁺ microglia in the SNpc (top) and in the striatum (bottom). Error bars represent ±SEM. (n = 5 SAL WT, n = 5 LPS WT, n = 6 SAL Snca^+/+, n = 6 LPS Snca^+/+; Ordinary two-way ANOVA for Genotype vs Treatment; SNpc: Interaction, F (1, 18) = 0.7841; Genotype, F (1, 18) = 0.7550; Treatment, F (1, 18) = 9.796, p = 0.0058; striatum: Interaction, F (1, 18) = 0.1473). d Mean fluorescence of CD86 both in the SNpc (top) and striatum (bottom). (n = 5 SAL WT, n = 5 LPS WT, n = 6 SAL Snca^+/+, n = 6 LPS Snca^+/+; Ordinary two-way ANOVA for Genotype vs Treatment; SNpc: Interaction, F (1, 18) = 2.858; Genotype, F (1, 18) = 79.88, p < 0.0001; Treatment, F (1, 18) = 9.063, p = 0.0075; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, with Bonferroni’s post hoc multiple comparisons test; striatum: Interaction, F (1, 18) = 4.848, p = 0.0410; Genotype, F (1, 18) = 111.9, p < 0.0001; Treatment, F (1, 18) = 25.47, p < 0.0001; **p < 0.01, ***p < 0.001, ****p < 0.0001, with Bonferroni’s post hoc multiple comparisons test). e Mean fluorescence of MHC-II both in the SNpc (top) and striatum (bottom). (n = 5 SAL WT, n = 5 LPS WT, n = 6 SAL Snca^+/+, n = 6 LPS Snca^+/+; Ordinary two-way ANOVA for Genotype vs Treatment; SNpc: Interaction, F (1, 18) = 0.2921; Genotype, F (1, 18) = 56.27, p < 0.0001; Treatment, F (1, 18) = 623.2, p < 0.0001; ***p < 0.001, ****p < 0.0001, with Bonferroni’s post hoc multiple comparisons test; striatum: Interaction, F (1, 18) = 7.674, p = 0.0126; Genotype, F (1, 18) = 24.36, p = 0.0001; Treatment, F (1, 18) = 40.04, p < 0.0001; ***p < 0.001, ****p < 0.0001, with Bonferroni’s post hoc multiple comparisons test).

Fig. 2. Peripheral immune cells into the SN and the striatum 3 months after LPS administration.

a Bar plots of the percentage of CD45^highCD3⁺ T cells in the SNpc (top) and the striatum (bottom). (n = 5 SAL WT, n = 5 LPS WT, n = 6 SAL Snca^+/+, n = 6 LPS Snca^+/+; Ordinary two-way ANOVA for Genotype vs Treatment; SNpc: Interaction, F (1, 18) = 11.58, p = 0.0032; Genotype, F (1, 18) = 25.14, p < 0.0001; Treatment, F (1, 18) = 14.07, p = 0.0015; ***p < 0.001, ****p < 0.0001, with Bonferroni’s post hoc multiple comparisons test; striatum: Interaction, F (1, 18) = 2.041; Genotype, F (1, 18) = 15.05, p = 0.0011; Treatment, F (1, 18) = 44.63, p < 0.0001; *p < 0.05, **p < 0.01, ****p < 0.0001, with Bonferroni’s post hoc multiple comparisons test). b Bar plots of the percentage of CD45^highCD45RA⁺ B cells in the SNpc (top) and the striatum (bottom). (n = 5 SAL WT, n = 5 LPS WT, n⁼6 SAL Snca^+/+, n = 6 LPS Snca^+/+; Ordinary two-way ANOVA for Genotype vs Treatment; SNpc: Interaction, F (1, 18) = 0.02012; striatum: Interaction, F (1, 18) = 1.916). c Bar plots of the percentage of CD45^highCD161⁺NK cells in the SNpc (top), while, in the striatum (bottom), a significant genotype effect in Snca^+/+ compared to WT groups is shown, independent from treatment. (n = 5 SAL WT, n = 5 LPS WT, n = 6 SAL Snca^+/+, n = 6 LPS Snca^+/+; Ordinary wo-way ANOVA for Genotype vs. Treatment; SNpc: Interaction F (1, 18) = 0.01421; striatum: Interaction, F (1, 18) = 0.01596; Genotype, F (1, 18) = 33.17, p < 0.0001; Treatment, F (1, 18) = 2.033; *p < 0.05, **p < 0.01, ***p < 0.001, with Bonferroni’s post hoc multiple comparisons test).

Fig. 3. LPS accelerates alpha synuclein accumulation in the SN of Snca^+/+ rats.

a Left, representative immunostaining of pSyn (S129) in the SNpc (top, scale: 200 µm) and related micrograph (bottom, scale: 50 μm). Right, densitometric quantification of pSyn (S129) in SNpc. Each point represents mean O.D. values per animal ±S.E.M. (n = 5 rats/group; Kruskal-Wallis test: p = 0.0011 (approximate), **p = 0.0079, with Mann Whitney test). b Left, representative immunostaining of pSyn (S129) in the VTA (top, scale: 200 µm) and micrograph (bottom, scale: 50μm). Right, densitometric quantification of pSyn (S129) in VTA. Each point represents mean O.D. values per animal ±S.E.M. (n = 5 rats/group; Ordinary Two-way ANOVA for Genotype vs Treatment: Interaction, F (1, 16) = 0.01289; Genotype, F (1, 16) = 138.5, ****p < 0.0001; Treatment, F (1, 16) = 0.3907). c Left, representative immunostaining of pSyn (S129) in the striatum (scale: 50μm) and densitometric quantification, right. Each point represents mean O.D. values per animal ±S.E.M. (n = 5 rats/group; Ordinary Two-way ANOVA for Genotype vs. Treatment: Interaction, F (1, 15) = 5.632, p = 0.0314; Genotype, F (1, 15) = 73.16, p < 0.0001; Treatment, F (1, 15) = 7.504, p = 0.0152; *p = 0.122, with Bonferroni’s post hoc multiple comparisons test). d Left, representative immunostaining of pSyn (S129) in the NP (scale: 50 μm) and densitometric quantification, right. Each point represents mean O.D. values per animal ±S.E.M. (n = 5 rats/group; Ordinary Two-way ANOVA for Genotype vs Treatment: Interaction, F (1, 15) = 0.03596).

Fig. 4. Morphological alterations in SNpc induced by LPS.

a Representative coronal images midbrain, SN, and VTA slices from 3,3’-diaminobenzidine (DAB)-stained TH⁺ neurons in WT and Snca^+/+ rats treated with saline (SAL) or Lipopolysaccharide (LPS) at 3 months after injection (scale: 200 µm, 100 µm, 20 µm). b Unbiased stereological TH⁺ cell count plot in the SNpc. Each point represents actual values ± SEM. (n = 5 rats/group; Kruskal-Wallis test: p = 0.0031 (approximate); *p = 0,0159, **p = 0,0079, with Mann Whitney test). c Unbiased stereological TH⁺ cell count plot in the VTA. Each point represents actual values ± SEM. (n = 5 rats/group; (n = 5 rats/group; Ordinary two-way ANOVA for Genotype vs Treatment: Interaction, F (1, 15) = 0.3154). d Representative confocal images of stained SNpc sections for TH (green) and DDC (red) dopaminergic markers and NeuroTrace (NT, blue) in WT and Snca^+/+ rats treated with saline (SAL) or Lipopolysaccharide (LPS) at 3 months after injection (scale: 20 µm). White arrowheads show TH^-/DDC⁺/NT⁺ neurons (magenta). e Unbiased stereological count of total TH⁺/DDC⁺ neurons in SNpc. Each point represents actual values ± SEM. (n = 5 rats/group; Kruskal-Wallis test: p = 0.0082 (approximate); **p < 0.01, with Mann Whitney test). f Unbiased stereological count of total TH^-/DDC^- neurons in SNpc. Each point represents actual values ± SEM (n = 5 rats/group; Kruskal-Wallis test: p = 0.9016 (approximate)).

Fig. 5. Morphological alterations in SNpr induced by LPS in Snca^+/+ rats.

a Representative image of coronal midbrain slice from DAB-stained SNpr TH⁺ fibers in WT and Snca^+/+ rats treated with SAL or LPS (scale: 50 µm) and related micrographs (scale: 10 µm). Black arrowheads show swollen TH⁺ dendrites morphology. b SNpr TH⁺ fibers densitometric analysis. Values are expressed as means of each animal normalized to the total mean value of SAL WT group ± SEM. (n = 5 rats/group; Ordinary two-way ANOVA for Genotype vs Treatment: Interaction, F (1, 16) = 6.614, p = 0.0205; Genotype, F (1, 16) = 8.053, p = 0.0119; Treatment, F (1, 16) = 6.497, p = 0.0214; *p < 0.05, **p < 0.01, with Bonferroni’s post hoc multiple comparisons test).

Fig. 6. LPS induced functional and morphological alterations in the striatum.

a Left, schematic representation showing the placement of the stimulating (blue arrowhead) and carbon fiber (white arrowhead) used for the recordings in the dorsal striatum. Middle, representative traces (scale: 100 pA; 500 ms) and evoked DA concentration in the dorsal striatum at 3 months after treatment in WT and Snca^+/+ rats recorded with constant potential amperometry. Right, bar plot showing max evoked DA outflow in Snca^+/+ rats. (Ordinary two-way ANOVA Genotype vs Treatment; Interaction, F (1, 343) = 2.395; Genotype, F (1, 343) = 41.03, p < 0.0001; Treatment, F (1, 343) = 5.763, p = 0.0169; *p < 0.05, **p < 0.01, ****p < 0.0001, with Tukey’s post hoc multiple comparisons test). b Left, representative traces showing the effects of cocaine at different concentrations (top: 0.3 μM; Bottom: 1 μM) in SAL (left) and LPS (right) Snca^+/+ rats. At the top right, the bar plot indicates the effect of cocaine on DA overflow (n = 4 SAL Snca^+/+ cocaine [0.3 μM], n = 3 LPS Snca^+/+ cocaine [0.3 μM], n = 3 SAL Snca^+/+ cocaine [1 μM], n = 3 LPS Snca^+/+ cocaine [1 μM]; Ordinary two-way ANOVA Treatment vs Cocaine concentrations; Interaction, F (1, 9) = 0.3939). On the right bottom, the bar plot indicates the effect of cocaine on the decay phase of the DAergic signal. Data are presented as mean ± SEM. (n = 4 SAL Snca^+/+ cocaine [0.3 μM], n = 3 LPS Snca^+/+ cocaine [0.3 μM], n = 3 SAL Snca^+/+ cocaine [1 μM], n = 3 LPS Snca^+/+ cocaine [1 μM]; Ordinary two-way ANOVA Treatment vs Cocaine concentrations; Interaction, F (1, 9) = 0.1171). c The bar plot shows the change in striatal DA release upon 5 min superfusion of quinpirole. Data are presented as mean ± SEM. (n = 3 rats/group; t-test, t = 0.1126). d Representative images of TH immunoreactivity of striatal coronal sections in WT and Snca^+/+ rats treated with SAL or LPS 3 months after treatment (scale bar: 500 μm). The values are expressed as means of each animal normalized to the total mean value of SAL WT group ± SEM. (n = 5 rats/group; Ordinary two-way ANOVA for Genotype vs Treatment; Interaction, F (1, 15) = 0.2409).