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, 15 (9), 1866-75

Inefficient DNA Repair Is an Aging-Related Modifier of Parkinson's Disease

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Inefficient DNA Repair Is an Aging-Related Modifier of Parkinson's Disease

Sara Sepe et al. Cell Rep.

Abstract

The underlying relation between Parkinson's disease (PD) etiopathology and its major risk factor, aging, is largely unknown. In light of the causative link between genome stability and aging, we investigate a possible nexus between DNA damage accumulation, aging, and PD by assessing aging-related DNA repair pathways in laboratory animal models and humans. We demonstrate that dermal fibroblasts from PD patients display flawed nucleotide excision repair (NER) capacity and that Ercc1 mutant mice with mildly compromised NER exhibit typical PD-like pathological alterations, including decreased striatal dopaminergic innervation, increased phospho-synuclein levels, and defects in mitochondrial respiration. Ercc1 mouse mutants are also more sensitive to the prototypical PD toxin MPTP, and their transcriptomic landscape shares important similarities with that of PD patients. Our results demonstrate that specific defects in DNA repair impact the dopaminergic system and are associated with human PD pathology and might therefore constitute an age-related risk factor for PD.

Figures

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Figure 1
Figure 1
NER Is Essential for the Integrity of the DA System (A) Targeted deletion of the key NER gene Ercc1 in DA neurons results in degeneration of striatal DA processes already evident in 26-week-old mice; DA neurons (red) and the general neuronal population (green) were revealed with anti-TH and anti-NeuN antibodies, respectively. Inset shows clear reduction of TH+ neurons in mutants. (B) Loss of nigral DA neurons is paralleled by reduced TH immunoreactivity in mutants’ striatum. Bar graphs illustrate quantification of striatal TH immunoreactivity. (C) Reduced TH+ neurons in SNpc and decreased striatal TH immunoreactivity i is exacerbated at 52 weeks of age. The scale bar represents 200 μm (A), 500 μm (B), and 1,000 μm (C). Error bars on the graph denote SEM. Six 26- and 52-week-old animals from each genotype were used for each experiment.
Figure 2
Figure 2
Alterations in the Nigro-striatal DA System in Ercc1Δ/+ Mice (A) Decreased striatal DA innervation in the caudate putamen (CPu) of Ercc1Δ/+ mice is paralleled by increased TH expression in SNpc neuronal bodies. Graphs on the right side represent the quantification of the fluorescent signal. (B) Increased oxidation in the SS/SH redox couple in SNpc DA neurons. The montage on the right side represents a zoom showing how the Metamorph software automatically defines regions of interest (ROIs) by using the TH signal as a mask. ROIs are then transferred to the image expressing the SS/SH ratio. (C) Ercc1Δ/+ mutants feature increased DNA damage as indicated by the higher proportion of DA neurons containing more than five γH2AX foci per nucleus. The scale bar represents 500 μm (A) and 150 μm (B). p < 0.05; ∗∗p < 0.01; unpaired t test. Error bars on the graph denote SEM. Six 20-week-old animals from each genotype were used in each experiment.
Figure 3
Figure 3
Ercc1Δ/+ Mice Display Hallmarks of PD Pathology and Share Similarities with PD Patients (A) Increased ubiquitination in the SNpc and in its DA neurons. The corresponding graph expresses intensity of the ubiquitin signal in TH+ regions of the image. (B) Increased phosphorylation of α-syn at ser129 (p-syn) in both cell bodies (arrows) and processes (arrowheads) of SNpc DA neurons. Graph expresses intensity of the p-syn signal in TH+ regions. (C) Impaired C-I-driven mitochondrial respiration in Ercc1Δ/+ mice. When energized with glutamate/malate, mitochondria extracted from the VM region of Ercc1Δ/+ mice exhibit decreased respiration in both state 4 and state 3. (D) Conversely, succinate-stimulated respiration is increased in both state 4 and state 3 in mitochondria extracted from the VM region of Ercc1Δ/+ animals. (E) Ultrastructural electron microscopy reveals abnormal mitochondria with disorganized cristae in Ercc1Δ/+ SNpc DA neurons. DA neurons are unambiguously identified by precipitated electron-dense DAB resulting from pre-embedding TH immunostaining. ER, endoplasmic reticulum; M, mitochondria. (F) RNA-seq and subsequent canonical pathway analysis reveals that pathways highly relevant for PD pathogenesis are altered in Ercc1Δ/+ VM. (G) Heatmap comparing downregulated (green) and upregulated (red) pathways in Ercc1Δ/+ mice, in PD, and in ILBD. (H) Venn diagrams showing the number of common downregulated and upregulated pathways in Ercc1Δ/+, PD, and human ILBD. The scale bar represents 15 μm (A and B) and 0.6 μm (C). p < 0.05; ∗∗∗p < 0.001; ∗∗p < 0.01; unpaired t test. Error bars on the graph denote SEM. Six (A, B, and E) to eight (C and D) 20-week-old animals for each genotype were used in each experiment.
Figure 4
Figure 4
Increased Sensitivity of Ercc1Δ/+ Mice to MPTP and Inefficient NER in PD Patients’ Fibroblasts (A) Decrease in striatal DA innervation induced by MPTP treatment is more pronounced in Ercc1Δ/+ mice. (B) Unbiased stereological counts indicating more severe MPTP-induced DA loss in Ercc1Δ/+ mice. The scale bar represents 500 μm (A) and 200 μm (B). p < 0.05; two-way ANOVA. Error bars on the graph denote SEM. For the experiment, five 20-week-old animals for each group were used. (C) Mild NER defects in fibroblasts derived from idiopathic (iPD; n = 11) and genetic (LRRK2-PD; n = 4) PD patients, but not from AD cases, as measured by UDS assay. Genetic PD cells harbor either the G2019S (n = 2) or the R1441G (n = 2) mutation. p < 0.05; ∗∗p < 0.01; one way ANOVA. No significant differences were detected in cells derived from AD patients (n = 4). Box and whiskers representation also shows the mean of the values (+). (D) DSB repair efficiency was evaluated in PD and AD fibroblasts. PD cases did not differ from control cases 1 hr after exposure to gamma irradiation (2 Gy), whereas they exhibited a significant increase in the number γH2AX foci still present 24 hr after irradiation (left graph), which suggests impaired DSB repair capacity. AD cases did not present significant differences from respective, matched controls p < 0.05; two-way ANOVA. Error bars on graph represent SEM.

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