Mitochondrial damage-associated inflammation highlights biomarkers in PRKN/PINK1 parkinsonism

Abstract

There is increasing evidence for a role of inflammation in Parkinson's disease. Recent research in murine models suggests that parkin and PINK1 deficiency leads to impaired mitophagy, which causes the release of mitochondrial DNA (mtDNA), thereby triggering inflammation. Specifically, the CGAS (cyclic GMP-AMP synthase)-STING (stimulator of interferon genes) pathway mitigates activation of the innate immune system, quantifiable as increased interleukin-6 (IL6) levels. However, the role of IL6 and circulating cell-free mtDNA in unaffected and affected individuals harbouring mutations in PRKN/PINK1 and idiopathic Parkinson's disease patients remain elusive. We investigated IL6, C-reactive protein, and circulating cell-free mtDNA in serum of 245 participants in two cohorts from tertiary movement disorder centres. We performed a hypothesis-driven rank-based statistical approach adjusting for multiple testing. We detected (i) elevated IL6 levels in patients with biallelic PRKN/PINK1 mutations compared to healthy control subjects in a German cohort, supporting the concept of a role for inflammation in PRKN/PINK1-linked Parkinson's disease. In addition, the comparison of patients with biallelic and heterozygous mutations in PRKN/PINK1 suggests a gene dosage effect. The differences in IL6 levels were validated in a second independent Italian cohort; (ii) a correlation between IL6 levels and disease duration in carriers of PRKN/PINK1 mutations, while no such association was observed for idiopathic Parkinson's disease patients. These results highlight the potential of IL6 as progression marker in Parkinson's disease due to PRKN/PINK1 mutations; (iii) increased circulating cell-free mtDNA serum levels in both patients with biallelic or with heterozygous PRKN/PINK1 mutations compared to idiopathic Parkinson's disease, which is in line with previous findings in murine models. By contrast, circulating cell-free mtDNA concentrations in unaffected heterozygous carriers of PRKN/PINK1 mutations were comparable to control levels; and (iv) that circulating cell-free mtDNA levels have good predictive potential to discriminate between idiopathic Parkinson's disease and Parkinson's disease linked to heterozygous PRKN/PINK1 mutations, providing functional evidence for a role of heterozygous mutations in PRKN or PINK1 as Parkinson's disease risk factor. Taken together, our study further implicates inflammation due to impaired mitophagy and subsequent mtDNA release in the pathogenesis of PRKN/PINK1-linked Parkinson's disease. In individuals carrying mutations in PRKN/PINK1, IL6 and circulating cell-free mtDNA levels may serve as markers of Parkinson's disease state and progression, respectively. Finally, our study suggests that targeting the immune system with anti-inflammatory medication holds the potential to influence the disease course of Parkinson's disease, at least in this subset of patients.

Keywords: IL6; PINK1; Parkinson’s disease; ccf-mtDNA; parkin.

Figure 1

Interleukin 6 (IL6) is increased in serum from biallelic PRKN/PINK1 mutation carriers compared to healthy controls. IL6 levels in serum samples from Parkinson’s disease (PD) patients with mutations in PRKN/PINK1, unaffected mutation carriers, idiopathic Parkinson’s disease (IPD) patients, and healthy control subjects (HC). (A) Patients with Parkinson’s disease due to biallelic PRKN/PINK1 mutations (mut+/+ PD+, n = 15) exhibited higher IL6 levels compared to healthy controls (n = 84). Unaffected heterozygous mutation carriers (mut+/− PD−, n = 13) showed similar IL6 levels compared to affected heterozygous mutation carriers (mut+/− PD+, n = 18). (B) Investigating an independent cohort from Italy yielded similar group differences as presented in A, while the total amount of IL6 among all participants was lower compared to the German cohort due to a batch effect (mut+/+ PD+, n = 19; mut+/− PD+, n = 5; mut+/− PD−, n = 9; HC, n = 9). Regarding the Italian cohort, biallelic PRKN/PINK1 mutation carriers exhibited increased IL6 levels compared to unaffected heterozygotes. Moreover, there was a trend towards elevated IL6 levels in PRKN/PINK1 affected heterozygotes compared to healthy control subjects. (C) In the German cohort, PRKN/PINK1 biallelic patients showed a trend towards higher IL6 levels compared to patients with idiopathic Parkinson’s disease (n = 51), while idiopathic Parkinson’s disease patients exhibited a trend towards elevated IL6 release compared to healthy control subjects. As multiple testing was performed, the significance level was adjusted to α = 0.0167. The assumed order among the different groups was tested with the Jonckheere-Terpstra test (bold, lower left corner). Pairwise differences between two groups were assessed using the Wilcoxon rank sum test. Data are presented as box and whisker plots. The box extends from the 25th to the 75th percentile. The line in the middle of the box represents the median. Tukey whiskers are used.

Figure 2

Serum ccf-mtDNA is increased in PRKN/PINK1 mutation carriers and discriminates between affected heterozygotes and idiopathic Parkinson’s disease patients. (A) Patients with Parkinson’s disease (PD) due to biallelic PRKN/PINK1 mutations (mut+/+ PD+, n = 13) and affected heterozygous individuals (mut+/− PD+, n = 17) exhibited elevated serum ccf-mtDNA levels compared to idiopathic Parkinson’s disease patients (IPD, n = 57). Additionally, affected heterozygous patients showed higher ccf-mtDNA levels than healthy control subjects (HC, n = 55). Unaffected heterozygous mutation carriers (mut+/− PD−, n = 14) exhibited ccf-mtDNA levels between those of affected mutation carriers and idiopathic Parkinson’s disease patients. As multiple testing was performed, the significance level was adjusted to α = 0.0167. Group differences in general were tested via Kruskal-Wallis test (bold). Pairwise differences between two groups were analysed using the Wilcoxon rank sum test. Data are presented as box and whisker plots. The box extends from the 25th to the 75th percentile. The line in the middle of the box represents the median. Tukey whiskers are used. (B) Assessment of the utility of ccf-mtDNA levels to discriminate between idiopathic Parkinson’s disease patients (n = 57) and affected heterozygous PRKN/PINK1 mutation carriers (n = 17) investigated by a receiver operator characteristic (ROC) curve.

Figure 3

Association between age at examination, disease duration and IL6 release. (A) In patients with idiopathic Parkinson’s disease (n = 51), we observed elevated IL6 levels with increasing age when determining the Spearman’s correlation of both variables. (B) In PRKN/PINK1 biallelic and heterozygous patients (n = 31), there was a positive correlation between disease duration and IL6 levels. The Spearman’s correlation was calculated after adjusting for age at examination. Correlation coefficient is shown in the top left, exploratory P-values are presented in the top right corner. Lines represent linear regression.