An empty E1, E3, E4 adenovirus vector protects photoreceptors from light-induced degeneration

Abstract

We have previously identified a neuroprotective effect associated with empty (E1(-), E3(-), E4(-)) adenovirus vector delivery in a model of light-induced, photoreceptor cell death. In this study, we further characterize this protective effect in light-injured retina and investigate its molecular basis. Dark-adapted BALB/c mice, aged 6-8 weeks, were exposed to standardized, intense fluorescent light for 96 or 144 h. Prior to dark adaptation, all mice received intravitreous injection of 1 x 10(9) particles of an empty (E1(-), E3(-), E4(-)) adenovirus vector in one eye and vehicle in the other. Following light challenge of 96 or 144 h, histopathological analysis and quantitative photoreceptor cell counts were conducted. Semiquantitative assessment of messenger ribonucleic acid (mRNA) for the apoptosis related genes: p50, p65, IkBa, caspase-1, caspase-3, Bad, c-Jun, Bax, Bak, Bcl-2, c-Fos, and p53 using quantitative reverse transcriptase polymerase chain reaction was performed on eyes following 12 h of light exposure. Following 96 h of light exposure, the photoreceptor cell density for E1(-), E3(-), E4(-) adenovirus vector and vehicle-injected eyes were 87.5 +/- 9.5 and 79.3 +/- 10.1, respectively, (p = 0.79). After 144 h of light exposure, the photoreceptor cell density was preserved in vector-injected eyes as compared to vehicle treated eyes, 68.9 +/- 10.0 and 49.2 +/- 4.6, respectively (p = 0.016). Relative mRNA levels of c-Fos and c-Jun at 12-h light exposure after injection differed significantly between vector- and vehicle-injected eyes (p = 0.036, 0.016, respectively). The expression of the other apoptosis-related genes evaluated was not significantly affected. This study investigates the molecular basis of photoreceptor neuroprotective pathway induction associated with E1(-), E3(-), E4(-) adenovirus vectors. The results indicate that empty adenovirus vectors protect photoreceptors from light-induced degeneration by the modulation of apoptotic pathways. Gene expression changes suggest that the suppression of c-Fos and c-Jun upregulation contributes significantly to the neuroprotective effect. Understanding the molecular basis of the neuroprotective pathway induction in photoreceptors is critical to the development of novel therapies for retinal degenerations.

Keywords: Adenoviral vector; Apoptosis; Neuroprotection; Null effect; Retinal light damage.

Fig. 1

Light micrographs of the treated and untreated BALB/c retinas following light exposure. Mice received no injection (a), intravitreous injection of vehicle (b, c), or 1 × 10⁹ viral particles of empty AdV vector (d, e). Three days after injection, the mice were exposed to continuous fluorescent light (2,500 lux) for 0 (a), 96 (b, d), and 144 h (c, e). Eyes injected with empty AdV showed moderate protection of photoreceptor cells (d, e) when compared to vehicle-injected eyes (b, c). GCL Ganglion cell layer, INL inner nuclear layer, ONL outer nuclear layer, RPE retinal pigment epithelial layer (Scale: bar = 20 μm)

Fig. 2

Morphometric analysis of nuclear cell count in ONL of light-exposed retina. Outer nuclear cell counts of vehicle-injected eyes with 0, 96, or 144 h of continuous light exposure (solid circles) were 108.8 ± 7.3, 79.3 ± 10.1, and 49.2 ± 4.6, respectively. Outer nuclear cell counts of AdV-injected eyes with 96 or 144 h of light exposure (open circles) were 87.5 ± 9.5 and 68.9 ± 10.0, respectively. After 144 h of light exposure, the photoreceptor cell density was preserved in vector-injected eyes as compared to vehicle treated. Asterisk, p = 0.016 (error bars = 1 SD)

Fig. 3

Analysis of c-Fos (a) and c-Jun (b) mRNA levels in retinas of BALB/c mice by qRT-PCR. Mice were either kept in darkness (black column) or exposed to 2,500 lux for 12 h with no injection (dark gray column), vehicle injection (light gray column), or AdV injection (open column). c-Fos and c-Jun mRNA expressions were reduced in retinas of vector-injected eyes when compared to retinas of vehicle-injected eyes (asterisk, p = 0.036, cross, p = 0.016)