Central and systemic IL-1 exacerbates neurodegeneration and motor symptoms in a model of Parkinson's disease

Abstract

Parkinson's disease is a neurodegenerative disorder with uncertain aetiology and ill-defined pathophysiology. Activated microglial cells in the substantia nigra (SN) are found in all animal models of Parkinson's disease and patients with the illness. Microglia may, however, have detrimental and protective functions in this disease. In this study, we tested the hypothesis that a sub-toxic dose of an inflammogen (lipopolysaccharide) can shift microglia to a pro-inflammatory state and exacerbate disease progression in an animal model of Parkinson's disease. Central lipopolysaccharide injection in a degenerating SN exacerbated neurodegeneration, accelerated and increased motor signs and shifted microglial activation towards a pro-inflammatory phenotype with increased interleukin-1beta (IL-1beta) secretion. Glucocorticoid treatment and specific IL-1 inhibition reversed these effects. Importantly, chronic systemic expression of IL-1 also exacerbated neurodegeneration and microglial activation in the SN. In vitro, IL-1 directly exacerbated 6-OHDA-triggered dopaminergic toxicity. In vivo, we found that nitric oxide was a downstream molecule of IL-1 action and partially responsible for the exacerbation of neurodegeneration observed. Thus, IL-1 exerts its exacerbating effect on degenerating dopaminergic neurons by direct and indirect mechanisms. This work demonstrates an unequivocal association between IL-1 overproduction and increased disease progression, pointing to inflammation as a risk factor for Parkinson's disease and suggesting that inflammation should be efficiently handled in patients to slow disease progression.

Fig. 1

Timeline for central or systemic stimulus. Experimental groups were divided into: (A–C) ‘Central stimulus’ with rats intrastriatally injected with 6-OHDA and LPS (6-OHDA/LPS) or vehicle (6-OHDA/Veh) at Day 12 into the SN. Rats intrastriatally injected with Vehicle and LPS (Veh/LPS) or vehicle (Veh/Veh) at day 12 into the SN (A). (B) The dexamethasone (DXM) group received a daily intraperitoneal dose of 10⁻⁸ M of DXM starting just after the LPS or vehicle stereotaxical injection or 10 mg/kg of S-methylisothiourea SMT 15 min prior to LPS or vehicle injection and daily after that. (C) In the IL1 activity blockade experiments, animals were injected with Ad IL-1ra or Ad β-gal in the MFB three days prior to 6-OHDA intrastriatal injection. (D) ‘Systemic stimulus’ with rats intrastriatally injected with 6-OHDA and intravenously with Ad IL1β (6-OHDA/Ad IL1β iv) or vehicle (6-OHDA/Ad β-gal iv) at Day 7 in the tail vein. Rats intrastriatally injected with Vehicle and intravenously with Ad IL1β (Veh/Ad IL1βiv) or Ad β-gal (Veh/Ad β-gal iv) in the tail vein.

Fig. 2

(A) Remaining number of TH positive cells in the SN 21 days after different central treatments. Quantitation of TH positive cells as a percentage of cells in the ipsilateral hemisphere versus the contralateral one. Two-way ANOVA (significant double interaction P < 0.05) was followed by Fisher's LSD post hoc test. ***P < 0.001 6-OHDA/LPS compared to 6-OHDA/Veh, Veh/LPS and Veh/Veh group. **P < 0.01 6-OHDA/Veh compared to Veh/LPS. ***P < 0.001 6-OHDA/Veh compared to Veh/Veh. Error bars represent SEM. (N = 7, 9, 5 and 4 for 6-OHDA/LPS, 6-OHDA/Veh, Veh/LPS and Veh/Veh groups, respectively). DXM group: Three-way ANOVA (significant triple interaction P < 0.01) was followed by Fisher's LSD post –hoc test. °°°P < 0.001 significant difference between 6-OHDA/LPS+DXM and 6-OHDA/LPS-DXM. °P < 0.05 significant difference between Veh/LPS+DXM and Veh/LPS-DXM. Error bars represent SEM. (N = 5, 4, 4 and 4 for 6-OHDA/LPS+DXM, 6-OHDA/Veh+DXM, Veh/LPS+DXM and Veh/Veh+DXM groups, respectively). SMT group: One-way ANOVA was followed by Fisher's LSD post hoc test. ^▪▪▪P < 0.001 significant difference between 6-OHDA/LPS+SMT and 6-OHDA/LPS-SMT. ^▪P < 0.05 significant difference between 6-OHDA/LPS+SMT and 6-OHDA/Veh+SMT. Error bars represent SEM. (N = 6, 4 and 5 for 6-OHDA/LPS+SMT, 6-OHDA/Veh+SMT and Veh/LPS+SMT groups, respectively). (B and C) Motor behavioural deficits after central treatments and the effects of anti-inflammatory agent on behavioural tests. (B) Cylinder test. The number of first paw placement on the surface of a cylinder was counted for each experimental group. Three-way ANOVA (significant striatum—paw double interaction P < 0.001) was followed by Fisher's LSD post hoc test. ***P < 0.001 when comparing 6-OHDA/LPS between left and right paws. **P < 0.01 when comparing 6-OHDA/Veh between left and right paws. Error bars represent SEM. (N = 9, 8, 12 and 8 for 6-OHDA/LPS, 6-OHDA/Veh, Veh/LPS and Veh/Veh groups, respectively). (C) Adjusting steps test at 15 and 20 days after 6-OHDA or Vehicle injection (pre-lesion measurements were carried out for every animal). Performance with the left paw (ipsilateral to the lesion) presented no difference in both directions (data not shown). Only performance with the right paw was impaired in both directions (backhand direction not shown). Three-way ANOVA (Striatum, SN and day with repeated measures for the last factor) was followed by Fisher's LSD post hoc test. °°P < 0.01, significant difference between 6-OHDA/LPS group and the pre-lesion measurement. **P < 0.01 significant difference among 6-OHDA/LPS group and other groups (6-OHDA/Veh, Veh/LPS and Veh/Veh) within the same time point. ^▵P < 0.05 difference among 6-OHDA/Veh group and other control groups (Veh/LPS and Veh/Veh) for Day 20. ^▪P < 0.05 significant difference between 6-OHDA/LPS group with or without DXM treatment at each time point (Day 15 and Day 20). Error bars represent SEM. (N = 9, 6, 12, 7, 5, 5 and 4 for 6-OHDA/LPS, 6-OHDA/Veh, Veh/LPS, Veh/Veh, 6-OHDA/LPS+DXM, 6-OHDA/Veh+DXM, Veh/LPS+DXM groups, respectively).

Fig. 3

Activation of microglial cells in the SN after different central treatments (A–I) DXM-treated groups are also shown (J–P). (A–C). Activation of microglial cells as demonstrated by GSA (green)/TH (red). (A). Animals injected with 6-OHDA/LPS mostly exhibited GSA + cells at stage 4. In addition stages 2–3 microglial cells can be observed surrounding the SNpc. The animals injected with 6-OHDA/Veh (B) and Veh/LPS (C) have GSA + cells at stages 2 and 3 in the SN. A′–A′′′: Examples of different stages of microglial activation magnified from A (yellow circles): A′, Stage 2 characteristic rod-shaped cell. Ramified processes can be visualized; A′′, Stage 3 amoeboid microglia with thick and stout processes; A′′′ Stage 4 Phagocytic cell, round-shaped body. (D–F). Activated microglia with macrophage characteristics confirmed by ED1 (green), in the SN labelled with TH (red) immunofluorescence. (D). ED1 + cells at stage 2, 3 and 4 are observed within the SN in the 6-OHDA/LPS group. However, control groups (6-OHDA/Veh and Veh/LPS) exhibited scarce ED1 labelling (E and F, respectively). (G–I). Expression of the class two major histocompatibility complex (MHC-II) (red) and rat IL-1β (rIL-1β−green) in the different treatments. (G). Stage 4 MHC-II + cells are observed within the SN but ramified MHC-II + cells are located surrounding the lesion in 6-OHDA/LPS group. Ramified rIL-1β + cells are also observed within the lesion in this group (G). (H) Animals treated with 6-OHDA/Veh showed MHC-II + cells at stage 2. No IL-1β label was observed in this group. (I) Some MHC-II + cells at stage 4 can be observed at the injection site in Veh/LPS group. Stages 2 and 3 cells were also observed surrounding the injection site. Ramified rIL-1β + cells can also be seen in the same region. (J–P) Microglial activation after Dexamethasone treatment. (J and K) Activation of microglial cells demonstrated by GSA (green)/in the SN labeled with TH (red) staining. (J) The animals injected with 6-OHDA/LPS and treated with DXM showed mostly stage 4 GSA + cells in the whole SN. However, the Veh/LPS DXM treated group exhibited stage 2–3 microglial cells in the SN (K). J′–J′′′: Examples of different stages of microglial activation magnified from J (yellow circles): J′, Stage 2 characteristic rod-shaped cell. Ramified processes can be visualized; J′′, Stage 3 amoeboid microglia with thick and stout processes; J′′′ Stage 4 Phagocytic cell, round-shaped body. Scale bar A′, A′′, A′′′ and J′, J′′, J′′′: 20 μm. (L and M) Activated microglia with macrophage characteristics confirmed by ED1(green) immunofluorescence. (L) 6-OHDA/LPS DXM treated group showed ED-1 + cells at stage 4 within the lesion site, but also exhibited microglial cells at stage 2 and 3 surrounding the SN. The control group, Veh/LPS treated with DXM mainly had ED-1 + cells at stages 2–3, but scarce cells at stage 4 can also be observed (M). (N–P). Expression of the major histocompatibility complex class two (MHC-II) (red) and rIL-1β (rat IL-1β−green) in DXM treatment. (N) The animals injected with 6-OHDA/LPS and DXM exhibited less MHC-II label than those observed in non-DXM-treated animals (G). Some cells reached stage 4, but most of them were at stages 2–3 (N). Ramified IL-1β + cells can also be observed within the lesion (N). (O) Stages 2 and 3 MHC-II + cells were observed in the SN of animals injected with Veh/LPS and DXM. No IL-1β label was observed in this group. Scale bar: 100 μm. (P) Number of MHC-II + cells at stage 4 in the SN of animals injected with 6-OHDA/LPS (N = 4), Veh/LPS (N = 4), 6-OHDA/LPS +DXM (N = 3) and Veh/LPS+DXM (N = 3). Two-way ANOVA (significant double interaction P < 0.05) followed by Fisher′s LSD post hoc test. **P < 0.01 6-OHDA/LPS+DXM compared to 6-OHDA/LPS-DXM. Error bars represent SEM.

Fig. 4

IL-1β expression measured by ELISA in the SN after different treatments. Two-way ANOVA (Striatum and SN) with significant double interaction P < 0.05 was followed by Fisher's LSD post hoc test. ***P < 0.001 6-OHDA/LPS (N = 10) compared to 6-OHDA/Veh (N = 10), Veh/LPS (N = 9) and Veh/Veh (N = 7). DXM group: IL-1 content in the SN by ELISA was also measured for Dexamethasone-treated animals (6-OHDA/LPS+DXM N = 5 and Veh/LPS+DXM N = 5). Two-way ANOVA (Striatum and DXM) with significant double interaction P < 0.05 was followed by Fisher's LSD post hoc test. P < 0.05 significant difference between 6-OHDA/LPS group with or without DXM treatment. Error bars represent SEM. SMT group: ELISA was also used to measure IL-1 in the SN of SMT-treated animals. (6-OHDA/LPS, 6-OHDA/Veh, Veh/LPS N = 6 for each group). After a one-way ANOVA no statistical difference was found between groups with or without SMT treatment. ***P < 0.001 6-OHDA/LPS+SMT compared to 6-OHDA/Veh+SMT and Veh/LPS +SMT. The minimal amount of IL-1 depicted in the graph represents the detection threshold of the assay.

Fig. 5

Inhibition of IL-1β activity. (A) Quantitation of remaining TH-positive cells as a percentage of cells in the ipsilateral hemisphere versus the contralateral one. Ad IL-1ra-treated rats: filled bars, Adβ-gal-treated rats: empty bars. Significant differences were analyzed with one-way ANOVA. ***P < 0.001 significant difference between 6OHDA/LPS group treated with Ad IL-1ra or with control adenovirus (Ad β-gal). (Ad IL-1ra/6-OHDA/LPS N = 6, Ad β-gal/6-OHDA/LPS N = 5, all other groups N = 4). Error bars represent SEM. (B–G) Microglial activation after AdIL-1ra treatment. (B and C) Microglial activation evidenced with GSA (green) in the SN labeled with TH (red). (B) Morphological activation at Stage 4 was observed in the SN of animals injected with 6-OHDA/LPS and treated with AdIL-1ra. Some stage 3 cells can be observed surrounding the SNpc. (C) Control animals injected with Veh/LPS and AdIL-1ra exhibited few GSA+ cells only at stages 2–3. (D and E). Phagocytic activity evidenced by ED-1 immunolabelling (green). (F and G). Expression of the class two major histocompatibility complex (MHC-II) (red) and rIL-1β (rat IL-1β–green) after AdIL-1ra treatment in 6-OHDA/LPS and Veh/LPS animals. (F) The SN of 6-OHDA/LPS + AdIL-1ra animals showed mostly stage 4 MHC-II+ cells. Ramified rIL-1β+cells were observed surrounding the lesion. (G) The SN of control animals exhibited a reduced number of MHC-II + and IL-1β+ cells at the injection site. Scale bar: 100 μm. B′, B′′, C′, C′′: Examples of different stages of microglial activation magnified from B and C (yellow circles): B′, Stage 3 amoeboid microglia with thick and stout processes; B′′, Stage 4 phagocytic cell, round-shaped body; C′, Stage 2 rod-shaped cell and C′′, Stage 3 amoeboid microglia. Scale bar B′, B′′, C′, C′′: 20 μm. (H) Quantitation of MHCII positive cells (stage 4) throughout the SN 21 days after 6-OHDA in animals with IL-1 activity inhibited (Ad IL-1ra – dark grey bars) or controls [Ad β-gal (light grey bars) or no Ad treatment (black bars)]. No statistically significant differences were found between the different treatments. (AdIL-1ra/6-OHDA/LPS N = 3, Ad β-gal/6-OHDA/LPS N = 3, AdIL-1ra/Veh/LPS N = 3, Ad β-gal/Veh/LPS N = 3). Error bars represent SEM.

Fig. 6

Systemic Ad IL-1β injection increased neuronal loss in the SN. (A) Remaining number of TH-positive cells in the SN after systemic injection. Quantitation of TH-positive cells as a percentage of cells in the ipsilateral hemisphere versus the contralateral one. Two-way ANOVA (Striatum and systemic stimulus) with significant double interaction P < 0.05 was followed by Fisher's LSD post hoc test. *P < 0.05, compare 6-OHDA/Ad IL-1βiv (N = 7) to 6-OHDA/Ad β-gal iv (N = 6). N = 9 and 8 for Veh/Ad IL-1βiv and Veh/Ad β-gal iv groups, respectively). Error bars represent SEM. (B and C) Microglial activation in the SN of animals injected with 6-OHDA/Ad IL-1β i.v. B. GSA-positive (B) and ED-1-positive (C) cells at stage 4 were observed in the SN of 6-OHDA/AdIL-1β i.v. Scale bar B: 50 μm. Scale bar C: 100 μm. Inset in B (B'): magnification showing a stage 4 activated microglial cell. Scale bar inset (B'): 10 μm. (D) Quantitation of MHCII positive cells throughout the SN after systemic challenge. Two-way ANOVA (Striatum and systemic stimulus) with significant double interaction P < 0.01 was followed by Fisher's LSD post hoc test. **P < 0.01 significant difference between 6-OHDA/Ad IL-1βiv compared to every other groups. N = 5, 4, 5 and 5 for 6-OHDA/Ad IL-1βiv 6-OHDA/Ad β-gal iv, Veh/Ad IL-1βiv and Veh/Ad β-gal iv groups, respectively. Error bars represent SEM.

Fig. 7

(A) Viability of SK-N-SH cells tested by MTT assay. Cells were pre-treated with 6-OHDA and subjected to escalating doses of IL-1. Concentration-dependent decreases in cell viability in response to IL-1 were observed. One-way ANOVA followed by Fisher's LSD post hoc test. **P < 0.01 significant difference between 3.125 μM 6-OHDA/0.0125 pg/μl of IL-1 and 3.125 μM 6-OHDA/0 pg/μl of IL-1. *P < 0.05 significant difference between 3.125 μM 6-OHDA/0.0625 pg/μl of IL-1 or 3.125 μM 6-OHDA/0.05 pg/μl of IL-1 and 3.125 μM 6-OHDA/0 pg/μl of IL-1. Every group was normalized to 100%. Error bars represent SEM. (B–I) iNOS expression and 3-NT immunohistochemistry in different animal groups. (B and C). Ramified iNOS and 3-NT positive cells were observed within the SN of 6-OHDA/LPS-treated animals. (D and E) 6-OHDA/Veh animal group was completely devoid of iNOS and 3-NT immunoreactivity. (F and G) Veh/LPS animals exhibited a reduced immunoreactivity of ramified iNOS positive cells at the injection site. No 3-NT label was observed in this group. (H and I) No iNOS expression or 3-NT immunoreactivity was observed in the 6-OHDA/LPS+SMT-treated group. Scale bar: 100 μm.