Role of Bax in death of uninfected retinal cells during murine cytomegalovirus retinitis

Abstract

Purpose: Extensive death of uninfected bystander neuronal cells is an important component of the pathogenesis of cytomegalovirus retinitis. Our previous results have shown that caspase 3-dependent and -independent pathways are involved in death of uninfected bystander cells during murine cytomegalovirus (MCMV) retinitis and also that Bcl-2, an important inhibitor of apoptosis via the Bax-mediated mitochondrial pathway, is downregulated during this process. The purpose of this study was to determine whether Bax-mediated mitochondrial damage has a significant role in the death of uninfected retinal cells.

Methods: BALB/c mice, Bax(-/-) mice, or Bax(+/+) mice were immunosuppressed with methylprednisolone and infected with 5 × 10(3) plaque-forming units (PFU) of the K181 strain of MCMV via the supraciliary route. Injected eyes were analyzed by plaque assay, electron microscopy, hematoxylin and eosin (H&E) staining, TUNEL assay, Western blot (for caspase 3, caspase 12, Bax, receptor interacting protein-1 [RIP1] and receptor interacting protein-3 [RIP3]), as well as immunohistochemical staining for MCMV early antigen and cleaved caspase 3.

Results: Significantly more Bax was detected in mitochondrial fractions of MCMV-infected eyes than in mitochondrial fractions of mock-infected control eyes. Furthermore, the level of cleaved caspase 3 was significantly lower in MCMV-infected Bax(-/-) eyes than in MCMV-infected Bax(+/+) eyes. However, more caspase 3-independent cell death of uninfected bystander retinal cells and more cleaved RIP1 were observed in Bax(-/-) than in Bax(+/+) eyes.

Conclusions: During MCMV retinitis, Bax is activated and has an important role in death of uninfected bystander retinal cells by caspase 3-dependent apoptosis. Although the exact mechanism remains to be deciphered, active Bax might also prevent death of some types of uninfected retinal cells by a caspase 3-independent pathway.

Keywords: Bax; apoptosis; murine cytomegalovirus; retinitis.

Figure 1

(A) Western blot showing Bax expression in the mitochondrial fraction from injected eyes of IS BALB/c mice, either mock-infected (MOCK) or MCMV-infected at day 7 after injection. (B) Ratio of Bax to Hsp60. Data are shown as mean ± SEM (n = 4) and compared by a 2-tailed t-test. **P < 0.01.

Figure 2

Titer of MCMV (Log10 ± SEM PFU/mL) in MCMV-injected eyes of IS Bax^+/+ and Bax^−/− mice at day 7 after injection. Data are shown as mean ± SEM (n = 4). Statistical analysis by 2-tailed t-test indicated no significant difference between the 2 groups.

Figure 3

(A–D) Representative photomicrographs of H&E staining in uninjected and MCMV-injected eyes of IS Bax^+/+ and Bax^−/− mice at day 7 after injection. (E) Scoring of retinitis in MCMV-injected eyes of IS Bax^+/+ and Bax^−/− mice at day 7 after injection. Data are shown as mean ± SEM (n = 5) and compared by a 2-tailed t-test. *P < 0.05.

Figure 4

(A) Western blot of cleaved caspase 3 in MOCK-injected and MCMV-injected eyes of IS Bax^+/+ and Bax^−/− mice at day 7 after injection. (B) Ratio of cleaved caspase 3 to β-actin. Data are shown as mean ± SEM (n = 4) and compared by ANOVA. *P < 0.05, Bax^+/+, MCMV versus Bax^×/×, MCMV. (C, D) Merged photomicrographs (×200) of staining for MCMV EA (green), cleaved caspase 3 (red), and DAPI (blue) in MCMV-injected eyes of an IS Bax^+/+ and an IS Bax^−/− mouse at day 7 after injection. (E, F) Merged photomicrographs (×200) of staining for MCMV EA (red), TUNEL (green), and DAPI (blue) in MCMV-injected eyes of an IS Bax^+/+ and an IS Bax^−/− mouse at day 7 after injection. (G) Quantification of the number of TUNEL-positive cells in retinas of MCMV-injected Bax^+/+ eyes and MCMV-injected Bax^−/− eyes at day 7 after injection. The average number of TUNEL-positive cells for all retinal sections of each eye was used to determine the mean number of TUNEL-positive cells. Data are shown as the mean ± SEM (n = 4) and compared by 2-tailed t-test. *P < 0.05.

Figure 5

(A, B) The EMs showing photoreceptors exhibiting typical features of apoptosis (nuclear shrinkage and strong chromatin condensation, arrows) in the outer nuclear layer of MCMV-injected eyes of an IS Bax^+/+ and an IS Bax^−/− mouse at day 7 after injection. A few photoreceptors with nuclear condensation also exhibited a marked loss of cytoplasm due to cell lysis (arrowhead). (C) Quantification of the number of photoreceptor cells characterized with apoptosis-like cell death in retinas of MCMV-injected Bax^+/+ and Bax^−/− eyes at day 7 after injection. Data are shown as mean ± SEM (n = 15 fields) and compared by a 2-tailed t-test. ***P < 0.001.

Figure 6

(A) Western blot of cleaved caspase 12 in MOCK-injected and MCMV-injected eyes of IS Bax^+/+ and Bax^−/− mice at day 7 after injection. (B) Ratio of cleaved caspase 12 to β-actin. Data are shown as mean ± SEM (n = 4) and compared by ANOVA. *P < 0.05, Bax^+/+, MCMV versus Bax^−/−, MCMV. (C) Western blot of RIP1 and RIP3 in MOCK-injected and MCMV-injected eyes of IS Bax^+/+ and Bax^−/− mice at day 7 after injection. The membrane was initially stained with anti-RIP1 and anti-β-actin and subsequently with anti-RIP3 after treatment with stripping buffer. (D) Ratio of cleaved RIP1 to β-actin. Data are shown as mean ± SEM (n = 4) and compared by ANOVA. *P < 0.05, Bax^+/+, MCMV versus Bax^−/−, MCMV. (E) Ratio of full length RIP1 to β-actin. Data are shown as mean ± SEM (n = 4) and compared by ANOVA. *P < 0.05, Bax^+/+, MCMV versus Bax^−/−, MCMV. (F) Ratio of full length RIP3 to β-actin. Data are shown as mean ± SEM (n = 4) and compared by ANOVA. *P < 0.05, Bax^+/+, MCMV versus Bax^−/−, MCMV.