Lack of interleukin-13 receptor α1 delays the loss of dopaminergic neurons during chronic stress

Abstract

Background: The majority of Parkinson's disease (PD) cases are sporadic and idiopathic suggesting that this neurodegenerative disorder is the result of both environmental and genetic factors. Stress and neuroinflammation are among the factors being investigated for their possible contributions to PD. Experiments in rodents showed that severe chronic stress can reduce the number of dopaminergic neurons in the substantia nigra pars compacta (SNc); the same cells that are lost in PD. These actions are at least in part mediated by increased oxidative stress. Here, we tested the hypothesis that the interleukin-13 receptor alpha 1 (IL-13Rα1), a cytokine receptor whose activation increases the vulnerability of dopaminergic neurons to oxidative damage, participates in the stress-dependent damage of these neurons.

Methods: Mice were subject to daily sessions of 8 h (acute) stress for 16 weeks (5 days a week), a procedure previously showed to induce loss of dopaminergic neurons in the SNc. The source and the kinetics of interleukin-13 (IL-13), the endogenous ligand of IL-13Rα1, were evaluated 0, 1, 3, 6, and 8 h and at 16 weeks of stress. Identification of IL-13 producing cell-type was performed by immunofluorescent and by in situ hybridization experiments. Markers of oxidative stress, microglia activation, and the number of dopaminergic neurons in IL-13Rα1 knock-out animals (Il13ra1 ^{Y/ -} ) and their wild-type littermates (Il13ra1 ^Y/+ ) were evaluated at 16 weeks of stress and at 20 weeks, following a 4 week non-stressed period and compared to non-stressed mice.

Results: IL-13 was expressed in microglial cells within the SN and in a fraction of the tyrosine hydroxylase-positive neurons in the SNc. IL-13 levels were elevated during daily stress and peaked at 6 h. 16 weeks of chronic restraint stress significantly reduced the number of SNc dopaminergic neurons in Il13ra1 ^Y/+ mice. Neuronal loss at 16 weeks was significantly lower in Il13ra1 ^Y/- mice. However, the loss of dopaminergic neurons measured at 20 weeks, after 4 weeks of non-stress following the 16 weeks of stress, was similar in Il13ra1 ^Y/+ and Il13ra1 ^Y/- mice.

Conclusions: IL-13, a cytokine previously demonstrated to increase the susceptibility of SNc dopaminergic neurons to oxidative stress, is elevated in the SN by restraint stress. Lack of IL-13Rα1 did not prevent nor halted but delayed neuronal loss in the mouse model of chronic restraint stress. IL-13/IL-13Rα1 may represent a target to reduce the rate of DA neuronal loss that can occur during severe chronic restraint stress.

Keywords: Interleukin; Microglia; Neuroinflammation; Oxidative stress; Parkinson’s disease; Stress.

Fig. 1

Experimental restraint stress paradigms. Schematic representation of stress regimen and experimental design. a Daily sessions of 8 h RS were performed from 8 AM to 4 PM. Levels of IL-13 transcripts were determined at 1, 3, 6, and 8 h and the neuroinflammatory response. b Weekly schedule of RS consisted 8 h RS/day for 5 consecutive days per week followed by 2 days of non-stress. c Severe chronic RS consisted of the weekly RS schedule for a maximum up to was applied for 16 weeks. Distinct groups of animals were maintained in non-stressed conditions for four additional weeks following last session of stress before tissue was collected for analysis

Fig. 2

Acute restraint stress induces central production of IL-13 in the substantia nigra. a Semi-quantitative RT-PCR performed on RNA extracted from the SN of mice treated with acute RS showed that compared with the level of control animals, arbitrarily fixed to 1 (n = 8), levels were 0.37 ± 0.032, p > 0.99; 0.90 ± 0.186, p > 0.99; 3.89 ± 1.545, p = 0.04 and 2.63 ± 0.704, p = 0.74 at 1, 3, 6, and 8 h, respectively, n = 6. b Semi-quantitative RT-PCR assessing expression of IL-13Rα1 on same samples described in figure A shows no difference in all time points compared to control condition (p > 0.99 at control, 1, 3, 6, and 8 h). c Protein quantification in the SN shows upregulation of IL-13 during restraint stress (IL-13 in pg/mg total protein: 2.644 ± 0.50 control; 5.607 ± 2.793 at 1 h RS, p > 0.99;7.128 ± 3.123, at 3 h, p = 0.679; 8.857 ± 1.361 at 6 h RS, p = 0.1612, and 11.7 ± 3.195 at 8 h RS, p = 0.0342)

Fig. 3

Mapping the location of IL-13 production in the substantia nigra. (A–A″) Representative pictures of immunofluorescent staining of IL-13 (green) in the SN of non-stressed (top), 8 h of RS (center) and after 16 weeks of RS (bottom); showing that RS elevates IL-13 expression in both the SNc and the SNr. (Pictures representative of a n = 3 experiment; scale bars: 0.5 mm; blue: DAPI). (B–B″, C–C″, D–D″, E–E″) Representative pictures of double immunofluorescence of IL-13 (green) with Iba-1, GFAP, NeuN, and TH (red), respectively, in control condition, 8 h of RS, and 16 weeks of RS. Arrows: co-localization of IL-13 and the specific cell type marker; arrowheads: IL-13 signal without co-localization with the cell type marker. IL-13 co-localizes (arrows) with microglia (Iba-1, Fig. B–B″), neurons (NeuN, Fig. D–D″), and dopaminergic cells (TH, Fig. E–E″). No co-staining was found in astrocytes (GFAP, Fig. C–C″). (scale bar: 200 μm in A–A″, 50 μm in B–E″; blue: DAPI)

Fig. 4

IL-13Rα1 does not alter restraint stress-associated neuroinflammation. a Representative images showing immunofluorescence of 3-Nitrotyrosine (3-NT, green) in the SN under resting conditions and at different paradigms of RS in IL-13Rα1 wild-type and knock-out animals. Levels of oxidative damage were increased during restraint stress with no difference between genotypes. b Histograms showing the average levels of fluorescence intensity (in a.u.) of 3-Nitrotyrosine fluorescence in the SN. c Representative pictures of microglial marker Iba-1 under resting conditions and at different paradigms of RS in IL-13Rα1 wild-type and knock-out animals. Levels of oxidative were increased during restraint stress with no difference between genotypes. d Histograms showing the average levels of O.D.s (in a.u.) of Iba-1 in the SN (*p < 0.05, ***p < 0.001 compared to control condition, n = 4–6; scale bars: 100 μm in A, 200 μm in C)

Fig. 5

IL-13Rα1 contributes to dopaminergic neuronal loss during chronic stress. a Representative images of labeling of TH+ neurons using wild-type Il13ra ^Y/+ and knock-out Il13ra ^Y/− mouse brains from control mice and at different time points in the RS paradigm. b Histograms showing the number of TH+ neurons in the Il13ra ^Y/+and Il13ra ^Y/− in the SNc under control conditions, RS 8 h, after 16 weeks of RS, and after 16 weeks of RS plus 4 weeks of rest. c Representative images of cresyl violet labeling (Nissl staining) cells used in adjacent section to the ones assessed by TH staining. d Histograms showing the average cell count of CV+ cells in the Il13ra ^Y/+ and Il13ra ^Y/− in the SNc under control conditions, RS 8 h, after 16 weeks of RS, and after 16 weeks of RS plus 4 weeks of rest. (*p < 0.05, ***p < 0.001 compared to control condition, n = 4–6; scale bars: 200 μm in A, C)