Sustained delivery of NT-3 from lens fiber cells in transgenic mice reveals specificity of neuroprotection in retinal degenerations

Abstract

Several neurotrophic factors (NTFs) are effective in protecting retinal photoreceptor cells from the damaging effects of constant light and slowing the rate of inherited photoreceptor degenerations. It is currently unclear whether, if continuously available, all NTFs can be protective for many or most retinal degenerations (RDs). We used transgenic mice that continuously overexpress the neurotrophin NT-3 from lens fibers under the control of the alphaA-crystallin promoter to test for neuroprotection in light-damage experiments and in four naturally occurring or transgenically induced RDs in mice. Lens-specific expression of NT-3 mRNA was demonstrated both by in situ hybridization in embryos and by reverse-transcriptase polymerase chain reaction (RT-PCR) in adult mice. Furthermore, NT-3 protein was found in abundance in the lens, ocular fluids, and retina by enzyme-linked immunosorbent assay (ELISA) and immunocytochemistry. Overexpression of NT-3 had no adverse effects on the structure or function of the retina for up to at least 14 months of age. Mice expressing the NT-3 transgene were protected from the damaging effects of constant light to a much greater degree than those receiving bolus injections of NT-3. When the NT-3 transgene was transferred into rd/rd, Rds/+, Q344ter mutant rhodopsin or Mertk knockout mice, overexpression of NT-3 had no protective effect on the RDs in these mice. Thus, specificity of the neuroprotective effect of NT-3 is clearly demonstrated, and different molecular mechanisms are inferred to mediate the protective effect in light-induced and inherited RDs.

Figure 1

The NT-3 construct used to generate transgenic mice. The murine αA-crystallin promoter was linked to an 800 bp rat NT-3 cDNA followed by SV40 virus derived sequences including a 64 bp intron (represented by the line connecting the SV40-labeled box to the pA-labeled box) and polyadenylation site (SV40pA). Locations of primers used for genotyping and RT-PCR are indicated by labeled arrowheads.

Figure 2

Lens-specific transgene expression. Histological sections of E14.5 OVE613 Tg embryos were used for in situ hybridizations to riboprobes specific for the SV40 sequences present in the transgene. Hybridizations were done using an antisense riboprobe (A and B) and a sense riboprobe (C and D). Results are shown in bright-field (A and C) and corresponding dark-field (B and D) photomicroscopy. Transgene transcripts are indicated by silver grains. Hybridization signals were confined to the lens (L) with no hybridization detected in the neural retina (NR) or cornea (C). Non-Tg embryos showed no hybridization to either the antisense or sense riboprobes (data not shown). The melanin in the pigmented retinal pigment epithelium (RPE) appears bright in dark-field illumination (B and D). The lens sections demonstrate a non-specific glow with dark-field illumination that is unrelated to the hybridization signals (D). Hybridization signals were confined to the lens (L) with no hybridization detected in the neural retina (NR) or cornea (C). Scale bar = 200 µm.

Figure 3

NT-3 transgene expression in adult eyes. Ocular RNAs were amplified by RT-PCR using primers SV40A and SV40B. These primers amplify a 236 bp fragment from reverse transcribed mRNA in tissues expressing the transgene. The 236 bp fragment is present in reverse transcribed (RT +) lens RNA from the OVE613 and OVE614 transgenic lines (lanes 5 and 8). These primers amplify a band of 300 bp from transgenic genomic DNA (lane 3). No amplification is seen with nontransgenic FVB/N genomic DNA (lane 4). A 300 bp band, most likely representing contaminating genomic DNA, is amplified from lens RNA untreated with reverse transcriptase (RT -) (lanes 6 and 9) and from reverse transcribed total RNA isolated from the rest of the eye (ROE) (lanes 7 and 10).

Figure 4

ELISA measurements of NT-3 expression. NT-3 protein levels were assayed in the lenses, ocular fluids and retinas of Tg and non-Tg mice. The lenses of NT-3 Tg mice contain about 40 times as much NT-3 as non-Tg lenses. The ocular fluids (vitreous and aqueous humor) contain detectable NT-3 in the Tg but not non-Tg mice. The NT-3 content of the retinas in Tg mice is twice that of non-Tg mice. The lenses, retinas and ocular fluids (aqueous and vitreous humors combined) were harvested from 13 separate NT-3 Tg mice and 20 non-Tg mice at P70–75. The tissues from both eyes of each mouse were pooled so that the values shown are per two eyes. *P < 0.005; **P < 5 × 10⁻⁶; ***P < 5 × 10⁻¹⁹. The small amount of ocular fluid was difficult to collect, and in 7 of the 13 Tg animals the NT-3 level was below the limit of detection. If these 7 mice are omitted from the calculation, the value of the ocular fluids is 4.81 ± 3.07 ng/ml, and the statistical difference between the Tg and non-Tg mice is more significant (P < 5 × 10⁻⁷).

Figure 5

Immunocytochemical assays for NT-3. Cryostat sections of retinas (top row) and lenses (bottom row) were assayed for NT-3 protein (red fluorescence) A and F, NT-3 Tg mouse with normal retina at age P409. B and G, non-Tg BALB/c mouse with normal retina at P413. C, D, H and I, mertk^kd/+ mice, with normal retinas. C and H are non-Tg for NT-3, P150; D and I are Tg for NT-3, P180. E and J, mertk^kd/mertk^kd mouse, non-Tg for NT-3, with loss of photoreceptors at P180. Immunoreactivity with antibodies to NT-3 was present in the inner retina of both NT-3 Tg and non-Tg mice (A–E), and in the lenses of only the NT-3 Tg mice (F and I). The immunoreactivity in the lenses of Tg mice was present in the lens fibers (LF), but not in the lens epithelium (LE). In the retinas of NT-3 Tg mice (A and D), the intensity of immunoreactivity was greater than in the non-Tg mice (B, C and E). In both Tg and non-Tg retinas, NT-3 was detected primarily in the outer plexiform layer (OPL) and inner plexiform layer (IPL), with some signal present in neuronal cytoplasm in the inner nuclear layer (INL) and ganglion cell layer (GCL). Little or no signal was found in the outer nuclear layer (ONL) or among the photoreceptor inner segments, although the very small flecks of apparent immunoreactivity in the ONL in the Tg retinas (A and D) may represent Müller cell or PR localization. The apparent immunoreactivity of photoreceptor outer segments (OS) is due either to non-specific binding or autofluorescence, since it was still seen in control experiments in which the primary antibody was omitted (data not shown). Scale bar = 25 µm.

Figure 6

Representative ERG waveforms from non-Tg and NT-3 Tg mice. Scotopic (A) and photopic (B) waveforms from single mice at the age of P180 are shown. The ERG responses from the Tg mice were indistinguishable in amplitude and timing from the responses of wild-type littermates at this and earlier ages. Arrows indicate the time of stimulus onset.

Figure 7

Overexpression of NT-3 protects photoreceptors from constant light-induced degeneration. Light micrographs of the posterior retinas of NT-3 Tg mice either maintained in cyclic light (A) or exposed to constant light for 3 wks (B), and of a littermate non-Tg mouse exposed to 3 wks of constant light (C). A, The retina of the Tg mouse maintained in cyclic light is indistinguishable from that of wild-type, non-Tg mice. B, After 3 wks of constant light, the retina of the NT-3 Tg mouse shows the loss of most photoreceptor outer segments, but more than 50% of the photoreceptor nuclei in the outer nuclear layer (ONL) survived the light exposure. C, After 3 wks of constant light, the retina of the non-Tg mouse showed significantly greater damage than that of the NT-3 Tg mouse, with only a single row of photoreceptor nuclei surviving. Retinas from all were taken at P79; B and C, mice were placed into constant light at P58. IS, inner segments; OS, outer segments; RPE, retinal pigment epithelium. Scale bar = 25 µm.

Figure 8

Protection from constant light (CL) damage by Tg overexpression of NT-3 and intravitreally injected NT-3. Outer nuclear layer (ONL) thickness is a measure of photoreceptor number. A, NT-3 Tg mouse retinas show survival of significantly more photoreceptor nuclei than non-Tg littermates after 2 or 3 wks of CL. *P < 5 × 10⁻⁵; **P < 5 × 10⁻⁷ B, Protection by Tg expression compared to protection by intravitreal injection of NT-3. The data from panel A are expressed as “percent greater than control” in B. The NT-3 injected eyes (Inj) show significantly less protection from the damaging effect of CL than that seen in mice with Tg overexpression of NT-3 (P <0.005 at both 2 wk and 3 wk). The difference between the injected and control eyes was significant at both 2 wk and 3 wk (P < 0.005 and P <0.01, respectively). All mice were put into CL at P58, and the data are based on 6–7 mice in each group. The ONL thickness of wild-type mice at this age is 40–45 µm.

Figure 9

Overexpression of NT-3 fails to slow photoreceptor degeneration in 4 inherited models for retinal degeneration. NT-3 Tg mice were bred to rd/rd, Q344ter/+, Rds/+ and mertk^kd mice to provide retinal degeneration mutants either expressing the NT-3 transgene (Tg) or not (Non-Tg). The outer nuclear layer (ONL) thickness (mean ± SD) was determined at ages where protection would have been seen. No significant differences were found between the NT-3 Tg and non-Tg mice in any of the mutants. The ages and the number of mice quantified were the following: rd/rd, P20, n = 5 Tg and 5 non-Tg; Q344ter/+, P20, n = 5 Tg and 5 non-Tg; Rds/+, P55-P60, n = 9 Tg and 9 non-Tg; and mertk knockout, P40, n = 5 Tg and 7 non-Tg. As described in the Methods, the mean ONL thickness of each eye was based on 54 measurements around the eye, except for the Rds/+ mice, where 6 measurements were made in the peripheral 500 µm of both the superior and inferior hemispheres (i.e., 12 measurements from each eye). The mean ONL thickness of the peripheral retina of wild-type mice was 37.1 ± 2.9 µm.