Arginase 2 deficiency reduces hyperoxia-mediated retinal neurodegeneration through the regulation of polyamine metabolism

Abstract

Hyperoxia treatment has been known to induce neuronal and glial death in the developing central nervous system. Retinopathy of prematurity (ROP) is a devastating disease in premature infants and a major cause of childhood vision impairment. Studies indicate that, in addition to vascular injury, retinal neurons are also affected in ROP. Using an oxygen-induced retinopathy (OIR) mouse model for ROP, we have previously shown that deletion of the arginase 2 (A2) significantly reduced neuro-glial injury and improved retinal function. In the current study, we investigated the mechanism of A2 deficiency-mediated neuroprotection in the OIR retina. Hyperoxia treatment has been known to induce neuronal death in neonates. During the hyperoxia phase of OIR, a significant increase in the number of apoptotic cells was observed in the wild-type (WT) OIR retina compared with A2-deficient OIR. Mass spectrometric analysis showed alterations in polyamine metabolism in WT OIR retina. Further, increased expression level of spermine oxidase was observed in WT OIR retina, suggesting increased oxidation of polyamines in OIR retina. These changes were minimal in A2-deficient OIR retina. Treatment using the polyamine oxidase inhibitor, N, N'-bis (2, 3-butadienyl)-1, 4-butanediamine dihydrochloride, significantly improved neuronal survival during OIR treatment. Our data suggest that retinal arginase is involved in the hyperoxia-induced neuronal degeneration in the OIR model, through the regulation of polyamine metabolism.

Figure 1

Hyperoxia-induced cell death in the OIR retina. (a) Cell death was studied using TUNEL assay on cryostat sections. Numerous apoptotic cells were present in the WT OIR retina. In the A2^−/− OIR retina, fewer TUNEL-positive cells were observed compared with WT OIR retina. (b) Quantification of TUNEL-positive cells showed that the WT OIR retina had increased numbers of apoptotic cells at P8, P9 and P12 (1, 2 and 5 days of hyperoxia, respectively). However, this increase was not observed in the A2^−/− OIR retina. Data are presented as mean±s.d. *WT RA versus WT OIR (P<0.01), ^#WT RA versus WT OIR (P<0.05), ^§A2^−/− OIR versus WT OIR (P<0.01), N varies from four to six. Scale bar=50 μm. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer

Figure 2

Expression of arginase 2 (A2) in the OIR retina. (a and b) Immunostaining of retinal cryostat sections on postnatal day 9 showing immunoreactivity for A2 in horizontal cells and in the ONL in the WT RA and hyperoxia-treated WT OIR retina. (c) A2^−/− OIR retina used as negative control. Scale bar=50 μm. (d and e) High-magnification confocal images of WT RA and WT OIR retina showing immunolocalization of A2 in the horizontal cells and outer nuclear layer. Scale bar=20 μm. (f and g) High-magnification confocal images of A2 and DAPI showing A2 localization in the area of inner and outer segment development at P9. DAPI shows photoreceptor nuclei. Scale bar=20 μm. (h) Western blot analysis showing the expression of A2 in RA and OIR retinas. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer; OPL, outer plexiform layer; *Area of inner and outer segment development

Figure 3

Analysis of horizontal cells in the WT OIR retina. (a and b) Calbindin immunostaining of horizontal cells on RA and OIR sections. Scale bar=50 μm. (c) Quantification showing a significant decrease in the number of calbindin-positive horizontal cells in WT OIR retina compared with RA controls. *P<0.05, N=5. (d) Colocalization of TUNEL-positive cells with calbindin-positive horizontal cells in WT OIR retina. Scale bar=50 μm. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer

Figure 4

Analysis of polyamines in OIR retina. (a) Simplified flow chart showing the polyamine interconversion pathway. SMO, spermine oxidase; APAO, acetyl polyamine oxidase; SSAT, spermine spermidine acetyl transferase. Modified from Narayanan et al. (b) Using mass spectrometry, levels of spermine, spermidine, putrescine, N-acetyl spermine and N-acetyl spermidine were analyzed in the retinal samples collected on P9 (2 days of hyperoxia) and their relative levels are normalized to internal standards used. Changes in polyamine metabolism are observed in WT OIR retina. Data presented as mean±S.E.M. *WT RA versus WT OIR (P<0.05), ^§A2^−/− OIR versus WT OIR (P<0.05), N varies from 10 to 12

Figure 5

Increased polyamine oxidation in the OIR retina. (a) Western blot analysis showing increased expression of spermine oxidase (SMO) in WT OIR retina during different stages of hyperoxia. SMO expression is significantly reduced in A2^−/− OIR retina and is comparable to RA controls at all stages of hyperoxia studied. (b) Quantification of SMO expression in RA and OIR samples using ImageJ software. Data presented as mean±S.D. *WT RA versus WT OIR (P<0.01), ^§A2^−/− OIR versus WT OIR (P<0.01), N varies from 4 to 6. (c–n) Confocal images of SMO immunolocalization on retinal cryostat sections on postnatal day 9. Top panel (c–f) shows SMO expression in various retinal layers. Arrows represent areas of high expression. Scale bar=50 μm. Middle panel (g–j) shows increased expression of SMO in the OPL and ONL in WT OIR retina. Scale bar=20 μm. High-magnification confocal images of SMO and DAPI in the bottom panel (k–n) show SMO expression in the ONL and RPE cells. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer, *RPE. Scale bar=10 μm. (o) Analysis of hydrogen peroxide formation in the OIR retina using Amplex red showing increased H₂O₂ in WT OIR retina compared with A2^−/− OIR and room air controls. Data presented as mean±S.D. *WT RA versus WT OIR (P<0.01), ^§A2^−/− OIR versus WT OIR (P<0.01), N=5

Figure 6

Signaling mechanisms of hyperoxia-mediated neuronal death in OIR retina. (a) Western blot analysis showing the phosphorylation levels of JNK/SAPK P54 and JNK/SAPK P46. (b) Quantification of phosphorylation changes in JNK/SAPK P54 and JNK/SAPK 46. (c) Western blot analysis showing the expression of FASL, BID, cleaved BID and cytochrome c in WT OIR retina as compared with A2^−/− OIR retina and RA controls. (d) Quantification of changes in these signaling molecules in WT RA, WT OIR, A2^−/− RA and A2^−/− OIR retinas. Data are presented as mean±S.D. *WT RA versus WT OIR (P<0.01), ^§A2^−/− OIR versus WT OIR (P<0.01), N varies from four to six

Figure 7

Impact of polyamine oxidase inhibition in the OIR retina. (a) TUNEL assay performed on retinal sections from RA and OIR mice treated with vehicle or MDL 72527 from P7 to P9. (b) Quantification of the number of TUNEL-positive cells per retinal section in RA and OIR retina treated with vehicle or the polyamine oxidase inhibitor MDL 72527, showing significant neuronal survival in MDL 72527-treated OIR retina. (c) Measurement of H₂O₂ formation using Amplex Red assay in the RA and OIR retina treated with vehicle or MDL 72527. Data presented as mean±S.D. *Vehicle OIR versus MDL 72527 OIR (P<0.01), N varies from four to six. Scale bar=50 μm. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer

Figure 8

Effect of MDL 72527 treatment on apoptotic signaling in the OIR retina. (a) Western blot analysis showing changes in p-JNK/SAPK, t-JNK/SAPK FASL and cytochrome c with MDL 72527 treatments. (b) Quantification of changes in apoptotic signaling molecules in response to MDL 72527 treatment in the OIR retina. Data presented as mean±S.D. *Vehicle OIR versus MDL 72527 OIR (P<0.01), ^§Vehicle OIR versus MDL 72527 OIR (P<0.05), N=5