Both the neuronal and inducible isoforms contribute to upregulation of retinal nitric oxide synthase activity by brain-derived neurotrophic factor

Abstract

Although neurotrophins are best known for their trophic functions, growing evidence suggests that neurotrophins can also be neurotoxic, for instance by enhancing excitotoxic insults. We have shown recently that brain-derived neurotrophic factor (BDNF) limits its neuroprotective action on axotomized rat retinal ganglion cells (RGCs) by upregulating nitric oxide synthase (NOS) activity (Klöcker et al., 1998). The aim of the present study was to investigate this interaction of BDNF and NOS in the lesioned adult rat retina in more detail. We used NOS immunohistochemistry and NADPH-diaphorase (NADPH-d) reaction to characterize morphologically retinal NOS expression and activity. Using reverse transcription-PCR and Western blot analysis, we were able to identify the NOS isoforms being regulated. Six days after optic nerve lesion, we observed an increase in neuronal NOS (NOS-I) mRNA and protein expression in the inner retina. This did not lead to a marked increase in overall retinal NOS activity. Only RGC axons displayed strong de novo NADPH-d reactivity. In contrast, intraocular injection of BDNF resulted in a marked upregulation of NOS activity in NOS-I-immunoreactive structures, leaving the level of NOS-I expression unchanged. In addition, an induction of inducible NOS (NOS-II) was found after BDNF treatment. We identified microglial cells increasing in number and being activated by BDNF, which could serve as the cellular source of NOS-II. In summary, our data suggest that BDNF upregulates retinal NOS activity by both a post-translational regulation of NOS-I activity and an induction of NOS-II. These findings might be useful for developing pharmacological strategies to improve BDNF-mediated neuroprotection.

Fig. 1.

Effects of ON transection and BDNF on NOS histochemical activity in the adult rat retina using NADPH-d reactivity as a marker. Radial sections of untreated control retinas (A, C) and retinas 6 d after ON transection without treatment (B) and after a single intraocular injection of 500 ng of BDNF on day 4 after lesion (D). Sections were processed in parallel to allow comparison of NADPH-d reactivity. The arrow in B indicates RGC axons displaying NADPH-d reactivity after ON transection. Thethick arrow in D points to a type I amacrine neuron, and the thin arrow points to a type II amacrine neuron. ONL, Outer nuclear layer. Scale bar, 45 μm.

Fig. 2.

Effects of ON transection and BDNF on NOS histochemical activity in the nerve fiber layer of adult rat retinas using NADPH-d reactivity as a marker. Whole mounts of an untreated control retina (A) and retinas 6 d after ON transection without treatment (B) and after a single intraocular injection of 500 ng of BDNF on day 4 after lesion (C). Whole mounts in A–C were processed in parallel to allow comparison of NADPH-d reactivity. Note the increase in axonal NADPH-d reactivity after ON transection. Magnification: 100×. Close-up into the ganglion cell layer of an untreated control retina (D).Arrows indicate two presumptive displaced amacrine cells. Magnification: 400×.

Fig. 3.

Effect of BDNF on NOS histochemical activity in the INL of adult rat retinas using NADPH-d reactivity as a marker. Whole mounts of an untreated control retina (A) and a retina 6 d after ON transection treated with a single injection of 500 ng of BDNF on day 4 after lesion (B). The retinas were processed in parallel to allow comparison of NADPH-d reactivity. The arrowsindicate type I (white) and type II (black) amacrine neurons in the INL, respectively. Scale bar, 45 μm.

Fig. 4.

A, Quantification of the effect of BDNF on NOS histochemical activity in the INL. Cell counts of NADPH-d-positive type I and type II neurons were performed following the same protocol as described for RGC counts (see Materials and Methods). Numbers are expressed as percentage of the respective contralateral control retinas. Data from three different animals per experimental group were pooled. B, Quantification of the effect of axotomy on NOS immunoreactivity in the INL. Cell counts of NOS-I-immunoreactive type I and type II amacrine neurons in absolute numbers per retinal section. Data from at least five central sections per animal from three animals per experimental group were pooled.

Fig. 5.

Regulation of retinal NOS mRNA and protein expression by ON transection and BDNF. A, Representative RT-PCR for NOS-I, NOS-II, and G3PDH of total retinal tissues of untreated controls (CTRL), from retinas 6 d after ON transection without treatment (AXO), and with a single injection of vehicle (VEH) or 500 ng of BDNF (BDNF) on day 4 after lesion.B, Representative Western blot analysis for NOS-I and NOS-II of total retinal tissue of the respective experimental groups. Retinal tissues of four different animals per experimental group were pooled except for NOS-II protein analysis, which displays in each case (VEH and BDNF) test and contralateral control retina of the same animal.

Fig. 6.

Effects of ON transection and BDNF on the expression of NOS-I immunoreactivity in the adult rat retina. Radial sections of an untreated control retina (A) and control ON head (B), of a retina and ON head 6 d after ON transection (C, D), and a retina 6 d after ON transection treated with a single intraocular injection of 500 ng of BDNF (E). We were able to unequivocally identify RGCs that displayed NOS-I immunoreactivity (F), using retrograde fluorescent tracing (inset). See Results for detailed explanation of arrows. ONL, Outer nuclear layer. Scale bars: A, C,E, F, 45 μm. Magnification:B, D, 200×.

Fig. 7.

Memantine does not prevent the BDNF-induced increase in retinal NADPH-d reactivity. Radial sections of retinas 6 d after ON transection treated with a single intraocular injection of 500 ng of BDNF on day 4 after lesion (A,B). In B, the intraocular BDNF treatment was combined with systemic administration of memantine at a dose of 20 mg/kg body weight twice daily. Sections were processed in parallel to allow comparison of NADPH-d reactivity. ONL, Outer nuclear layer. Scale bar, 45 μm.

Fig. 8.

BDNF activates retinal microglia. Complement receptor-3 (Ox-42; left column) and MHC-II antigen (Ox-6; right column) immunoreactivity is shown in radial sections of untreated control retinas (A,B) and of retinas 6 d after ON transection either without treatment (C, D) or with a single injection of vehicle (E, F) or 500 ng of BDNF (G, H) on day 4 after lesion. ONL, Outer nuclear layer. Scale bar, 45 μm.