Oxidative Stress and Microglial Response in Retinitis Pigmentosa

Abstract

An imbalance between the production of reactive oxygen species (ROS) and anti-oxidant capacity results in oxidative injury to cellular components and molecules, which in turn disturbs the homeostasis of cells and organs. Although retinitis pigmentosa (RP) is a hereditary disease, non-genetic biological factors including oxidative stress also modulate or contribute to the disease progression. In animal models of RP, the degenerating retina exhibits marked oxidative damage in the nucleic acids, proteins, and lipids, and anti-oxidant treatments substantially suppress photoreceptor cell death and microgliosis. Although the mechanisms by which oxidative stress mediates retinal degeneration have not been fully elucidated, our group has shown that oxidative DNA damage and its defense system are key regulators of microglial activation and photoreceptor degeneration in RP. In this review, we summarize the current evidence regarding oxidative stress in animal models and patients with RP. The clinical efficacy of anti-oxidant treatments for RP has not been fully established. Nevertheless, elucidating key biological processes that underlie oxidative damage in RP will be pivotal to understanding the pathology and developing a potent anti-oxidant strategy that targets specific cell types or molecules under oxidative stress.

Keywords: microglia; oxidative DNA damage; oxidative stress; retinitis pigmentosa.

Figure 1

Imbalance between reactive oxygen species (ROS) and anti-oxidants impairs the function of macromolecules. ROS including superoxide (¹O₂), hydrogen peroxide (H₂O₂), and hydroxyl radicals (^•OH) are generated during oxidative phosphorylation in the mitochondria. Superoxide dismutase (SOD) catalyzes the ¹O₂ into oxygen (O₂) and H₂O_2. Catalase breaks down ^•OH into O₂ and water (H₂O). Glutathione peroxidase (GPx) catalyzes H₂O₂ into H₂O, with the conversion of glutathione (GSH) to its oxidized disulfide form (GSSG). An imbalance between ROS production (red box) and anti-oxidant capacity (green box) results in the accumulation of oxidative insults (lightning symbol) to the cellular lipids, proteins, and nucleic acids. ↑, increase.

Figure 2

The Figure is reproduced from [16] with copyright permission. Accumulation of oxidative DNA damage in retinitis pigmentosa (RP). (A,B) Hematoxylin and eosin (HE) staining and immunohistochemical staining of 8-oxo-7,8-dihydroguannine (8-oxoG) in the retina of rd10 mice (A) and Royal College of Surgeons (RCS) rats (B), two genetically different models of retinitis pigmentosa. Per staining, HCl pretreatment was used to denature the nuclear DNA, thereby enhancing the detection of 8-oxoG in the nuclear DNA. Note that 8-oxo-G accumulation is substantially increased in the outer nuclear layer (ONL) of RP mice. Scale bar, 50 μm. INL: inner nuclear layer. (C) Enzyme-linked immunosorbent assay for 8-oxo-dG in the vitreous of RP patients and controls (patients with idiopathic epiretinal membrane). The vitreous levels of 8-oxo-dG are increased in RP patients. * p = 0.0003

Figure 3

Oxidative DNA damage and its repair system. Oxidation of DNA occurs via two processes: one is the direct oxidation by reactive oxygen species (ROS) and the other is the incorporation of oxidized bases from the nucleotide pool. Oxidative insults are shown in lightning symbols. During DNA replication, 8-oxoG can mispair with adenine, resulting in the formation of somatic mutations. Mut T homolog-1 (MTH1) hydrolyzes oxidized purine nucleoside triphosphates, such as 8-oxo-7,8-dihydrro-2′-deoxyguanosine 5′-triphosphate (8-oxo-dGTP) and 1,2-dihydro-2-oxo-2′-deoxyadenosine 5′-triphosphate (2-oxo-dATP) in the nucleotide pool, thereby preventing the incorporation of oxidized nucleotides into DNA. On the one hand, 8-oxoG in DNA is excised by 8-oxoguanine DNA glycosylase-1 (OGG1). On the other hand, the Mut Y homolog (MUTYH) excises the mismatched adenine opposite 8-oxoG, and 2-oxoA opposite guanine. MUTYH can suppress mutagenesis caused by oxidative DNA damage; however, under severe oxidative DNA damage, MUTYH-mediated BER leads to accumulation of single-strand breaks (SSBs) in DNA. SSBs in the nuclear DNA activates poly(ADP-ribose) polymerase (PARP) and SSBs in mitochondrial DNA results in calpain activation through mitochondrial dysfunction, which in turn induces microgliosis and neuronal cell death during neurodegeneration, respectively.

Figure 4

The Figure is reproduced from [16] with copyright permission. Transgenic overexpression of human Mut T homolog-1 attenuates oxidative DNA damage and suppresses retinal degeneration. (A) Histological examination of rd10 mice, rd10 mice with hemizygous human MutT homolog-1 (hMTH1) expression (rd10; hMTH1-Tg^+/−), or rd10 mice with homozygous hMTH1 expression (rd10; hMTH1-Tg^+/+). Scale bar, 50 μm. INL: inner nuclear layer. ONL: outer nuclear layer. (B) Quantitative analysis of the number of photoreceptor cells in the ONL. Note that transgenic overexpression of hMTH1 significantly suppresses photoreceptor cell loss in rd10 mice. (C) Immunohistochemical staining of 8-oxoG in the retina of rd10, rd10; hMTH1-Tg^+/−, or rd10; hMTH1-Tg^+/+ mice. 8-oxo-G accumulation in rd10 mice is attenuated by transgenic hMTH1 expression. Scale bar, 50 μm. * p < 0.05. ** p < 0.01.

Figure 5

The Figure is reproduced from [59] with copyright permission. Oxidative DNA damage in microglia mediates retinal inflammation and degeneration through Mut Y homolog activation. (A–C) Immunohistochemical staining of microglial marker Iba-1 (A), hematoxylin and eosin staining (B), and peanut agglutin (PNA) staining, which labels the cone inner and outer segments (C) in rd10 mice or rd10 mice deficient for Mut Y homolog (Mutyh) (rd10; Mutyh^−/−). Note that microgliosis and rod as well as cone degeneration are suppressed by Mutyh deficiency. ONL: outer nuclear layer. Scale bar, 50 μm (A,B), 20 μm (C,D). Time-dependent changes of oxidative DNA damage in rd10 mice. Scattered 8-oxoG accumulation is observed at early phase of retinal degeneration (postnatal day 17: P17), and thereafter 8-oxo-G accumulation expands to the ONL diffusely at P21 in rd10 mice. Scale bar, 50 μm. (E) Part of scattered 8-oxo-G staining in the P17 rd10 retinas are colocalized with Iba-1-positive microglial cells, suggesting that 8-oxo-G accumulation in microglia precedes the peak of photoreceptor cell death in rd10 mice. Scale bar, 20 μm. (F) Immunohistochemical staining of single-strand DNA (ssDNA) to detect single-strand breaks (SSBs) and poly(ADP-ribose) (PAR) for the marker of poly(ADP-ribose) polymerase (PARP) activation in rd10 or rd10; Mutyh^−/− mice. Microglia in rd10 mice are associated with SSB formation and PARP activation, which is reversed by Mutyh deficiency. Scale bar, 20 μm.