NDRG2 suppression as a molecular hallmark of photoreceptor-specific cell death in the mouse retina

Abstract

Photoreceptor cell death is recognized as the key pathogenesis of retinal degeneration, but the molecular basis underlying photoreceptor-specific cell loss in retinal damaging conditions is virtually unknown. The N-myc downstream regulated gene (NDRG) family has recently been reported to regulate cell viability, in particular NDRG1 has been uncovered expression in photoreceptor cells. Accordingly, we herein examined the potential roles of NDRGs in mediating photoreceptor-specific cell loss in retinal damages. By using mouse models of retinal degeneration and the 661 W photoreceptor cell line, we showed that photoreceptor cells are indeed highly sensitive to light exposure and the related oxidative stress, and that photoreceptor cells are even selectively diminished by phototoxins of the alkylating agent N-Methyl-N-nitrosourea (MNU). Unexpectedly, we discovered that of all the NDRG family members, NDRG2, but not the originally hypothesized NDRG1 or other NDRG subtypes, was selectively expressed and specifically responded to retinal damaging conditions in photoreceptor cells. Furthermore, functional experiments proved that NDRG2 was essential for photoreceptor cell viability, which could be attributed to NDRG2 control of the photo-oxidative stress, and that it was the suppression of NDRG2 which led to photoreceptor cell loss in damaging conditions. More importantly, NDRG2 preservation contributed to photoreceptor-specific cell maintenance and retinal protection both in vitro and in vivo. Our findings revealed a previously unrecognized role of NDRG2 in mediating photoreceptor cell homeostasis and established for the first time the molecular hallmark of photoreceptor-specific cell death as NDRG2 suppression, shedding light on improved understanding and therapy of retinal degeneration.

Fig. 1. Responses to retinal damaging factors in retinal tissues and cells.

a, b Representative H&E staining of retinal tissues (a) and statistical analysis of thickness of the outer nuclear layer (ONL, representing photoreceptor cell bodies) (b). Scale bar = 20 μm. N = 4~5 per group. RPE retinal pigment epithelium, IS/OS inner segment/outer segment, OPL outer plexiform layer, INL inner nuclear layer, IPL inner plexiform layer, GCL ganglion cell layer, NF nerve fiber, MNU N-Methyl-N-nitrosourea. c, d Cell morphology (c) and viability analysis (d) by methyl thiazolyl tetrazolium (MTT) of the 661 W photoreceptor cell line. e, f Cell morphology (e) and viability analysis (f) by MTT of the retinal ganglion cell-5 (RGC-5) cell line. Scale bar = 5 μm. N = 3 per group. H₂O₂, hydrogen peroxide. Data represents mean ± SD. *P < 0.05

Fig. 2. Involvement of N-myc downstream regulated gene 1 (NDRG1) in retinal damages.

a Immunofluorescent staining of NDRG1 in the retinal tissue. Scale bar = 20 μm. b, c Quantitative real time polymerase chain reaction (qRT-PCR) analysis of the mRNA expression level (b) and western blot analysis of the protein expression level (c) of NDRG1 in the retinal tissue. d–g mRNA and protein expression levels of NDRG1 in the 661 W and RGC-5 cell lines. N = 3 per group. Data represents mean ± SD. *P < 0.05

Fig. 3. Expression of NDRG family members in retinal tissues and cells.

a qRT-PCR analysis of the mRNA expression levels of NDRG family members in the 661 W and RGC-5 cell lines. b Western blot analysis of the protein expression level of NDRG2 in the 661 W and RGC-5 cell lines. c Immunofluorescent staining of NDRG2 in the retinal tissue. Scale bar = 20 μm. N = 3 per group. Data represents mean ± SD. *P < 0.05

Fig. 4. Involvement of NDRG2–4 in retinal cell damages.

a, b qRT-PCR analysis of the mRNA expression level (a) and western blot analysis of the protein expression level (b) of NDRG2 in the 661 W and RGC-5 cell lines. c, d qRT-PCR analysis of the mRNA expression level (c) and western blot analysis of the protein expression level (d) of NDRG3 in the 661 W and RGC-5 cell lines. e, f qRT-PCR analysis of the mRNA expression level (e) and western blot analysis of the protein expression level (f) of NDRG4 in the 661 W and RGC-5 cell lines. g–i qRT-PCR analysis of the mRNA expression levels of NDRG2–4 in the 661 W and RGC-5 cell lines. N = 3 per group. Data represents mean ± SD. *P < 0.05

Fig. 5. Function of NDRG2 in maintaining photoreceptor cell viability and counteracting oxidative stress.

a, b qRT-PCR analysis of the mRNA expression level (a) and western blot analysis of the protein expression level (b) of NDRG2 in the 661 W cell line. 661 W cells were transduced with either green fluorescent protein (GFP)-based lentiviral vector for NDRG2 overexpression (the NDRG2 group) or the vacant vector (the GFP group). c, d qRT-PCR analysis of the mRNA expression level (c) and western blot analysis of the protein expression level (d) of NDRG2 in the 661 W cell line. 661 W cells were transfected with either a short heparin RNA (shRNA) for NDRG2 knockdown (the shNDRG2 group) or the scrambled shRNA (the scramble group). e-g qRT-PCR analysis of the mRNA expression levels of NDRG1, 3 and 4 in the 661 W cell line upon NDRG2 manipulations. h-j MTT analysis of cell viability of the 661 W cell line upon NDRG2 manipulations. k, l qRT-PCR analysis of the mRNA expression levels of the antioxidant master transcription factor Nuclear factor E2-related factor 2 (Nrf2), the regulated antioxidant genes Heme oxygenase-1 (Ho-1) and NAD(P)H:quinone oxidoreductase-1 (Nqo-1) in the 661 W cell line upon NDRG2 manipulations. N = 3 per group. Data represents mean ± SD. *P < 0.05

Fig. 6. Participation of NDRG2 in retinal photoreceptor cell loss and protection.

a Immunofluorescent staining of NDRG2 in the retinal tissue. Scale bar = 20 μm. b, c qRT-PCR analysis of the mRNA expression level (b) and western blot analysis of the protein expression level (c) of NDRG2 in the retinal tissue. N = 6~7 per group. d Immunofluorescent staining of NDRG2 in the retinal tissue. Scale bar = 20 μm. NAM, nicotinamide. e, f qRT-PCR analysis of the mRNA expression level (e) and western blot analysis of the protein expression level (f) of NDRG2 in the retinal tissue. N = 5 per group. Data represents mean ± SD. *P < 0.05