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. 2008 Oct 22;28(43):10825-34.
doi: 10.1523/JNEUROSCI.3001-08.2008.

Parkin deficiency increases vulnerability to inflammation-related nigral degeneration

Tamy C Frank-Cannon  1 , Thi Tran, Kelly A Ruhn, Terina N Martinez, John Hong, Marian Marvin, Meagan Hartley, Isaac Treviño, Daniel E O'Brien, Bradford Casey, Matthew S Goldberg, Malú G Tansey
Affiliations

Parkin deficiency increases vulnerability to inflammation-related nigral degeneration

Tamy C Frank-Cannon et al. J Neurosci. .

Abstract

The loss of nigral dopaminergic (DA) neurons in idiopathic Parkinson's disease (PD) is believed to result from interactions between genetic susceptibility and environmental factors. Evidence that inflammatory processes modulate PD risk comes from prospective studies that suggest that higher plasma concentrations of a number of proinflammatory cytokines correlate with an increased risk of developing PD and chronic nonsteroidal anti-inflammatory drug regimens reduce the incidence of PD. Although loss-of-function mutations in the parkin gene cause early-onset familial PD, Parkin-deficient (parkin-/-) mice do not display nigrostriatal pathway degeneration, suggesting that a genetic factor is not sufficient, and an environmental trigger may be needed to cause nigral DA neuron loss. To test the hypothesis that parkin-/- mice require an inflammatory stimulus to develop nigral DA neuron loss, low-dose lipopolysaccaride (LPS) was administered intraperitoneally for prolonged periods. Quantitative real-time PCR and immunofluorescence labeling of inflammatory markers indicated that this systemic LPS treatment regimen triggered persistent neuroinflammation in wild-type and parkin-/- mice. Although inflammatory and oxidative stress responses to the inflammation regimen did not differ significantly between the two genotypes, only parkin-/- mice displayed subtle fine-motor deficits and selective loss of DA neurons in substantia nigra. Therefore, our studies suggest that loss of Parkin function increases the vulnerability of nigral DA neurons to inflammation-related degeneration. This new model of nigral DA neuron loss may enable identification of early biomarkers of degeneration and aid in preclinical screening efforts to identify compounds that can halt or delay the progressive degeneration of the nigrostriatal pathway.

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Figures

Figure 1.
Figure 1.
Schematic of systemic LPS administration regimens and measurable outcomes. Wild-type and parkin−/− mice were given low-dose LPS or an equivalent volume of saline vehicle twice a week intraperitoneally for the indicated times. Locomotor behavior was evaluated before and during the course of treatment. Groups of animals were killed as indicated for biochemical and immunohistological analyses. IHC, Immunohistochemistry.
Figure 2.
Figure 2.
Fine-locomotor deficits in Parkin−/− mice exposed to prolonged, serial low-dose systemic LPS. A, No significant differences were detected between genotypes or treatment groups on accelerating rotarod, suggesting no general malaise. Bars represent mean ± SEM; n = 8 per group. B, parkin−/− mice display significantly prolonged time to cross on narrow beam walk after LPS treatment regimens. Asterisks indicate significant differences compared with saline-treated wild-type and LPS-treated wild-type group, whereas double asterisks indicate difference from all other groups. The triangle indicates a significant difference compared with LPS-treated wild-type animals only.
Figure 3.
Figure 3.
Parkin−/− mice display increased vulnerability to nigral DA neuron loss induced by repeated low-dose systemic LPS. A, Unbiased stereological analysis indicates that parkin−/− mice exposed to 3 months of low-dose systemic LPS followed by a 3 month wait and mice exposed to 6 months of low-dose systemic LPS display a significant reduction of TH-immunopositive neurons in the SNpc. The loss of TH-positive neurons in parkin−/− mice is reflected in the loss of NeuN-immunopositive neurons, confirming true neuronal loss. B, Unbiased stereological analysis indicates no loss of TH-positive or NeuN-positive neurons in the VTA in any of the groups. In A and B, error bars represent SEM, and the number of mice in each group is denoted in parentheses (n). Asterisks indicate significant differences compared with wild-type, saline-treated animals by three-way ANOVA followed by Tukey's HSD post hoc test at α = 0.05. C, Images of TH (purple) and NeuN (brown) immunohistochemistry from single coronal sections of wild-type mice in the 6 month treatment group compared with the parkin−/− mice in each of the exposure groups. The black arrowhead in high-magnification inset indicates a double-labeled TH/NeuN-immunopositive neuron, and the white arrowhead indicates a NeuN-only (nondopaminergic) neuron. Scale bars: low-magnification panels, 400 μm; high-magnification inset, 10 μm.
Figure 4.
Figure 4.
Repeated low-dose intraperitoneal LPS injections does not cause loss of striatal TH-immunopositive terminals or DA depletion in parkin−/− or wild-type mice. A, Densitometric analysis of striatal TH fiber density (see Materials and Methods) indicates no significant differences between genotypes or treatment groups. Bars represent mean ± SEM; n = 4 animals per group. Kruskal–Wallis analysis was performed to assess equality of means; genotype, p = 0.89; treatment, p = 0.60. B, Representative striatal sections stained for TH from mice in the 6 month treatment groups. C, No significant differences were found by two-way ANOVA between genotypes or treatment in the 6 month groups in the levels of DA and its metabolites in the striatum as measured by HPLC and electrochemical detection (dopamine, p = 0.50; DOPAC, p = 0.31; HVA, p = 0.19; 3-MT, p = 0.47; DA turnover, p = 0.14; n = 7–8 animals per group).
Figure 5.
Figure 5.
Neuroinflammation induced by repeated low-dose systemic LPS administration persists throughout treatment regimen in wild-type and Parkin−/− mice. A, After administration of intraperitoneal LPS for 3 months, increased immunoreactivity for the pan-microglial marker CD68 (red) is detectable in the ventral midbrain region (delineated by the presence of TH-positive DA neurons; green) compared with saline-treated animals. Scale bar, 400 μm. B, Quantification of neuroinflammation markers COX-1, TNF, CD45, and CD68 by QPCR in the ventral midbrain of mice treated with low-dose systemic LPS for 6 months. Brain regions are MB and CX. Asterisks indicate significant difference relative to wild-type saline-treated animals. Bars represent mean ± SEM; n = 3–4 per group. Three-way ANOVA was performed with Tukey's HSD post hoc at α = 0.05 (TNF: F (7,31) = 3.93, p = 0.007; CD45: F (7,31) = 5.1, p = 0.001; CD68: not significant, p = 0.17; Cox-1: not significant, p = 0.19). Detailed pairwise comparisons revealed the following significant differences: CD45-MB wild-type saline and Parkin−/− saline are different from wild-type LPS and Parkin−/− LPS. CD45-CX Parkin−/− saline is different from Parkin−/− LPS. TNF-MB wild-type saline is different from Parkin−/− LPS and wild-type LPS. TNF-CX Parkin−/− LPS is different from wild-type saline and wild-type LPS.
Figure 6.
Figure 6.
Oxidative stress responses to prolonged, serial administration of low-dose systemic LPS are not exacerbated in parkin−/− mice. Real-time QPCR analyses of microdissected midbrain tissue measured expression levels of Nrf2, HO-1, NQO1, iNOS, SOD1, and SOD2. Significant differences between levels of iNOS mRNA expression in saline-injected wild-type and saline-injected parkin−/− mice were detected in the 6 month treatment groups. Whereas SOD2 mRNA was significantly increased in parkin−/− LPS compared with wild-type saline, the LPS-induced increases in iNOS, Nrf2, and HO-1 mRNA were similar for both genotypes. Differences in NQO1 and SOD1 mRNA did not reach significance. Bars represent mean ± SEM; n = 3–5 per group. Statistical significance (indicated by asterisks) was determined by three-way ANOVA with Tukey's HSD post hoc at α = 0.05 (Nrf2: F (7,31) = 4.9, p = 0.002; HO-1: F (7,30) = 2.94, p = 0.02; NQO1: not significant, p = 0.44; iNOS: F (7,30) = 5.26, p = 0.001; SOD1: not significant, p = 0.19; SOD2: F (7,31) = 3.0, p = 0.02).
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