Nanoceria extend photoreceptor cell lifespan in tubby mice by modulation of apoptosis/survival signaling pathways

Abstract

Cerium oxide nanoparticles, nanoceria, are inorganic antioxidants that have catalytic activities which mimic those of the neuroprotective enzymes superoxide dismutase and catalase. We have previously shown that nanoceria preserve retinal morphology and prevent loss of retinal function in a rat light damage model. In this study, the homozygous tubby mutant mouse, which exhibits inherited early progressive cochlear and retinal degeneration, was used as a model to test the ability of nanoceria to slow the progression of retinal degeneration. Tubby mice were injected systemically, intracardially, with 20 μl of 1mM nanoceria in saline, at postnatal day 10 and subsequently at P20 and P30 whereas saline injected and uninjected wild type (or heterozygous tubby) served as injected and uninjected controls, respectively. Assays for retinal function, morphology and signaling pathway gene expression were performed on P34 mice. Our data demonstrate that nanoceria protect the retina by decreasing Reactive Oxygen Species (ROS), up-regulating the expression of neuroprotection-associated genes; down-regulating apoptosis signaling pathways and/or up-regulating survival signaling pathways to slow photoreceptor degeneration. These data suggest that nanoceria have significant potential as global agents for therapeutic treatment of inherited retinal degeneration and most types of ocular diseases.

Figure 1

Nanoceria inhibit loss of function in the tubby mouse retina as determined by electroretinography (ERG) at P34. Full field ERG assessment demonstrated that nanoceria treatment (tub/tub-CeO₂) improved rod function in tub/tub mice when compared to saline treatment (tub/tub-saline) (A). The image shown is the average amplitude of 4 eyes from each group. Serial intensity ERG of scotopic a-wave (B) and b-wave (C) demonstrated that both amplitudes were significantly increased in nanoceria treated tub/tub eyes compared to the saline injection group. N=4 in each group. * P<0.05; ** P<0.01; *** P<0.001

Figure 2

Nanoceria provide pan-retinal protection of tubby photoreceptor cells. Photomicrographs of H&E stained retinas in superior from tub/tub treated with saline (tub/tub-saline), or with CeO₂ (tub/tub-CeO₂) and tub/wt injected with saline (tub/wt-saline) (A). Quantitative histology (B) shows that the outer nuclear layer (ONL) thickness was greatly reduced in the tub/tub mice (tub/tub-saline) compared to the tub/wt but was less reduced in nanoceria treated tub/tub eyes (tub/tub-CeO₂). Data shown in (C) is the average of all the measurements in the same group. N=54 measurements in each group. Scale bar, 100µm. *** P<0.001

Figure 3

Nanoceria increase the expression of bFGF, FGFR, Ras, p-ERK in the tubby retina. Western blots (A) were used to evaluate expression levels of survival signaling pathway components. Densitometric analysis showed that the elevation of bFGF (B), FGFR (C), Ras (D) and p-ERK (E) are statistically significant and comparable to the tub/wt levels when standardized to actin. The level of ERK protein was unaffected by the tubby mutation and nanoceria treatment (A). Data shown are representative of 4 western blots (A) and the average (mean ± SEM) of 4 retinas for quantitation (B–E). *P<0.05; **P<0.01

Figure 4

Nanoceria increase the expression of antioxidant associated proteins in the tubby retina. Western blot data (A) and densitometric analysis showed that nanoceria treatment increased Nrf-2 (B), N-Nrf-2 (C) and Trx (D) expression in the tubby retina to levels equivalent to the tub/wt controls. Data shown are representatives of 4 western blots (A) and the average (mean ± SEM) of 4 retinas for quantitation (B–D). *P<0.05; **P<0.01

Figure 5

ROS are elevated in the tubby retina and decreased by nanoceria treatment. Intracellular ROS levels were detected with dihydroethidium (DHE) for superoxide (O₂⁻) (A, B, C) and 2’, 7’-dichlorodijhydro-fluorescein diacetate (DCFH-DA) for hydrogen peroxide (H₂O₂) (D, E, F) on fresh cryosections at P18. Both assays show increased levels of ROS in the tubby retina treated with saline (A, D) compared to the control retina (C, F) and decreased ROS in the retinas of nanoceria treated tubby mice (B, E). Data shown are representative of 4 retinas from each group. ONL, outer nuclear layer. Scale bar, 100µm, Nuclei are stained with DAPI.

Figure 6

Nanoceria decrease mRNA expression of caspase-8 and Bak 1 and the activity of caspases-9 and -3 in the tubby retina. Semi-quantitative RT-PCR (A) shows increased caspase-8 and Bak 1 mRNA levels with saline treatment and a decreased mRNA levels in the retinas of nanoceria treated tubby mice. Quantitative measurements by qRT-PCR showed that the reductions of the gene expression were significant (B & C). Assays for caspases-9 (D) & -3 (E) show that they are elevated in saline treated tubby retinas and decreased in the nanoceria treated mice. Data are from 4 retinas from each group and representative data are shown in (A) and the averages (mean ± SEM) in (B-E). *P<0.05; **P<0.01

Figure 7

Nanoceria decrease indicators of apoptosis in the tubby retina. Levels of total and cytoplasmic cytochrome c are increased in the tubby retina (A, B, C) compared to the tub/wt control retina and nanoceria treatment decrease both. The TUNEL assay (D) shows similar changes with tub/tub (a) having many more TUNEL positive nuclei than the tub/wt retina (c) whereas treatment of tubby with nanoceria reduces these numbers. Data shown (A, D) are representative of those from 4 retinas from each group and their average (mean ± SEM) (B, C). *P<0.05, Scale bar, 40µm.

Figure 8

Documented changes in signal pathways in the tubby retina induced by nanoceria. The arrows within the title box indicate the changes in tubby retinas compared to wild type. The arrows in the colored boxes indicate nanoceria induced changes, either up or down, in the tubby retina. Neuroprotective genes are up regulated and pro-apoptosis genes are down-regulated by nanoceria. We think all of these nanoceria-induced changes are due to ROS downregulation and are indirectly regulated by nanoceria.