Parkinson's disease is associated with oxidative damage to cytoplasmic DNA and RNA in substantia nigra neurons

Abstract

Oxidative damage, including modification of nucleic acids, may contribute to dopaminergic neurodegeneration in the substantia nigra (SN) of patients with Parkinson's disease (PD). To investigate the extent and distribution of nucleic acid oxidative damage in these vulnerable dopaminergic neurons, we immunohistochemically characterized a common product of nucleic acid oxidation, 8-hydroxyguanosine (8OHG). In PD patients, cytoplasmic 8OHG immunoreactivity was intense in neurons of the SN, and present to a lesser extent in neurons of the nucleus raphe dorsalis and oculomotor nucleus, and occasionally in glia. The proportion of 8OHG immunoreactive SN neurons was significantly greater in PD patients compared to age-matched controls. Midbrain sections from patients with multiple system atrophy-Parkinsonian type (MSA-P) and dementia with Lewy bodies (DLB) also were examined. These showed increased cytoplasmic 8OHG immunoreactivity in SN neurons in both MSA-P and DLB compared to controls; however, the proportion of positive neurons was significantly less than in PD patients. The regional distribution of 8OHG immunoreactive neurons within the SN corresponded to the distribution of neurodegeneration for these three diseases. Nuclear 8OHG immunoreactivity was not observed in any individual. The type of cytoplasmic nucleic acid responsible for 8OHG immunoreactivity was analyzed by preincubating midbrain sections from PD patients with RNase, DNase, or both enzymes. 8OHG immunoreactivity was substantially diminished by either RNase or DNase, and completely ablated by both enzymes. These results suggest that oxidative damage to cytoplasmic nucleic acid is selectively increased in midbrain, especially the SN, of PD patients and much less so in MSA-P and DLB patients. Moreover, oxidative damage to nucleic acid is largely restricted to cytoplasm with both RNA and mitochondrial DNA as targets.

Figure 1.

8OHG immunoreactivity in midbrain structures from patients with Parkinson’s disease. 8OHG immunoreactivity is virtually undetectable in age-matched control brain sections (A), while it is abundant in the cytoplasm of SN neurons from PD patients (B). The brown pigments are neuromelanin. In some patients with PD, cytoplasmic 8OHG immunoreactivity was present in midbrain neurons and glia (arrow) outside of the SN (C). Scale bars: A and B, 10 μm; C, 25 μm.

Figure 2.

Quantitative Assessment of 8OHG Immunoreactivity. Data are 8OHG immunoreactive SN neurons expressed as percent of total neurons (mean ± SEM). *: P < 0.01 for control versus PD, MSA-P or DLB. ⁺: P < 0.05 for PD versus MSA-P or DLB.

Figure 3.

Effects of Purified 8OHG, DNase and RNase on 8OHG Immunoreactivity. Preabsorption of anti-8OHG with purified 8OHG completely ablated 8OHG immunoreactivity (A). 8OHG immunoreactivity in the SN from PD patients also was completely abolished by pretreatment of tissue with combined DNase and Rnase (B). Scale bar = 25 μm.