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. 2018 Jan 31;8(1):1968.
doi: 10.1038/s41598-018-20171-0.

Cone Degeneration Is Triggered by the Absence of USH1 Proteins but Prevented by Antioxidant Treatments

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Free PMC article

Cone Degeneration Is Triggered by the Absence of USH1 Proteins but Prevented by Antioxidant Treatments

Alix Trouillet et al. Sci Rep. .
Free PMC article

Abstract

Usher syndrome type 1 (USH1) is a major cause of inherited deafness and blindness in humans. The eye disorder is often referred to as retinitis pigmentosa, which is characterized by a secondary cone degeneration following the rod loss. The development of treatments to prevent retinal degeneration has been hampered by the lack of clear evidence for retinal degeneration in mutant mice deficient for the Ush1 genes, which instead faithfully mimic the hearing deficit. We show that, under normal housing conditions, Ush1g-/- and Ush1c-/- albino mice have dysfunctional cone photoreceptors whereas pigmented knockout animals have normal photoreceptors. The key involvement of oxidative stress in photoreceptor apoptosis and the ensued retinal gliosis were further confirmed by their prevention when the mutant mice are reared under darkness and/or supplemented with antioxidants. The primary degeneration of cone photoreceptors contrasts with the typical forms of retinitis pigmentosa. Altogether, we propose that oxidative stress probably accounts for the high clinical heterogeneity among USH1 siblings, which also unveils potential targets for blindness prevention.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Absence of a retinal phenotype in pigmented (C57Bl/6J genetic background) Ush1c−/− and Ush1g−/− mice. (ad) Electroretinogram (ERG) measurements performed at month 12 on wild-type control (grey), Ush1g−/− (red) and Ush1c−/− (blue) C57Bl/6 J mice showing no significant difference in the amplitudes of ERG responses, regardless of the test conditions: dark-adapted (a and b), photopic (c) or flickers (d). (e,f) OCT scans reveal no difference in retinal layers thickness of C57Bl/6 J Ush1g−/− mice (f) compared to control mice (e). In the retina of Ush1g−/− and Ush1c−/− C57Bl/6 J mice, the immunolabeling for cone blue opsin is well restricted to unaffected outer segments (h,i), resembling those observed in control mice (g). The scale bars represent 50 µm in (e and f), and 10 µm in (gi).
Figure 2
Figure 2
Cone dysfunction in albino Ush1c−/− and Ush1g−/− BALB/cJ mice. (a) ERG recordings in three-month-old (mo 3) and nine-month-old (mo 9) Ush1g−/− (red) and control (grey) BALB/cJ mice under dark-adapted (0.2 cds/m2), photopic and flicker (10 Hz) conditions. (b,c) Quantification of dark-adapted a-wave (rod hyperpolarization) and b-wave (bipolar neuron depolarization) ERG amplitudes (b), photopic ERG amplitudes and flicker amplitudes (c, cone pathway) at mo 3 and mo 9, showing significantly lower amplitudes in the mutant mice on photopic ERGs (mo 3: p = 0.025, n = 8; mo 9: p = 0.0011, n = 10), flickers (mo 3: p = 0.0012; mo 9: p = 0.0023) and dark-adapted ERG b-wave amplitudes (mo 9: p < 0.05). (d) ERG recordings in Ush1c−/− (blue) and control (grey) BALB/cJ mice at mo 9 under dark-adapted, photopic and flicker conditions. (e,f) Quantification of a-wave and b-wave amplitudes on dark-adapted (0.2 cds/m2) ERGs (e), amplitudes on photopic ERG and flicker ERGs (10 Hz) (f) at mo 3 and mo 9, showing significantly lower amplitudes of dark-adapted b-wave (p = 0.0036, n = 8) and photopic ERG (p = 0.0011, n = 7) responses in mutant mice at mo 9. The data shown are means ± SEM. (*), (**), and (***) denote p < 0.05, p < 0.01, and p < 0.005, respectively (Student’s t-test).
Figure 3
Figure 3
Mislocalization of cone opsins in Ush1c−/− and Ush1g−/− BALB/cJ mice and cone loss in Ush1g−/− BALB/cJ mice. (ac) Immunolabeling of M opsin on retinal cross-sections at month 3 (mo 3), showing that the outer segments (OS) are normal in shape in wild-type (a) and Ush1g−/− BALB/cJ mice (b), whereas the outer segments in Ush1c−/− BALB/cJ mice are disorganized (c, white arrowhead). (dg) The S opsin immunolabeling, which was restricted to the outer segment in wild-type BALB/cJ mice (d) revealed alterations to the outer segments in the two mutant mice, with mislocalized labeling extending over the entire photoreceptor cell body (white arrowheads, e and f). (g) Quantification of cone cells with mislocalized immunolabeling for S opsin (number of cells/mm retinal cross-section, p < 0.0001, Student’s t-test, n = 10 in each group). (hj) Lectin PNA staining on retinal cross-sections in 11-month-old control (h) and Ush1g−/− (i) BALB/cJ mice showing a reduction in the number of PNA-stained cone photoreceptor inner/outer segments in Ush1g−/− (red) BALB/cJ mice (j, number of cells/mm retinal cross-section, p < 0.05 n = 4 control animals, n = 7 Ush1g−/− mice). The scale bars represent 5 µm in (ac), 10 µm in (df) and 50 µm in (h and i). ONL: outer nuclear layer, INL: inner nuclear layer, IPL: inner plexiform layer.
Figure 4
Figure 4
Photoreceptor apoptosis in Ush1g−/− BALB/cJ mice. (ad) TUNEL staining in wild-type (a) and Ush1g−/− (bd) BALB/cJ mice at 3 mo. Apoptotic cells (labeled in red) are detected in the outer nuclear layer (ONL) of Ush1g−/− BALB/cJ retina (bd), whereas such cells were completely absent from the retina of control BALB/cJ mice (a). Note the apposition of the TUNEL labeling and the mislocalized S opsin immunolabeling (white arrowhead, d). The scale bars represent 50 µm in (a and b), 20 µm in (c), and 15 µm (d). OS: outer segment, INL: inner nuclear layer, IPL: inner plexiform layer.
Figure 5
Figure 5
GFAP and Retinal gliosis in Ush1c−/− and Ush1g−/− BALB/cJ mice. (ac) GFAP immunolabeling on retinal cross-sections display similar distribution patterns between control (a), Ush1g−/− (b) and Ush1c−/− (c) BALB/cJ mice. (dh) Iba1-immunopositive microglial cells on retinal sections. Microglial cells are regularly distributed in the inner (IPL) and outer (OPL) plexiform layers in control animals (d). In Ush1g−/− (e) and Ush1c−/− (f) BALB/cJ mice, activated microglial cells are also present in the regions of outer (OS) and inner (IS) segments of photoreceptor cells (white arrowhead, e). In (f), a microglial cell with a process extending in the outer nuclear layer (ONL) (white arrowhead). (g and h) Flat-mounted retinas of wild-type (g) and Ush1g−/− (h) BALB/cJ mice. The bulging and multiplication of cell bodies shown in the IPL and OPL of Ush1g−/− mice (h) is a feature of activated microglial cells. (i) Quantification on total flat-mounted retinas, comparing Ush1g−/− mice with age-matched controls, showed an increase in the numbers of microglial cells in the IPL, OPL, and throughout the entire retina. The data shown are means ± SEM. *p < 0.05, **p < 0.01 (Student’s t-test). The scale bars represent 15 µm in (af) and 50 µm (g and h). RPE: retinal pigment epithelium cell, INL: inner nuclear layer.
Figure 6
Figure 6
Darkness and taurine supplementation prevent retinal degeneration in Ush1-deficient mice. (a,b) Being kept in the dark for four months (from mo 2 to mo 6) prevented the occurrence of Ush1 abnormal phenotype in Ush1g−/− BALB/cJ mice (dark red graphs), as indicated by the lack of significant difference in photopic and flicker ERG responses, the normal localization of blue opsin in the outer segment (OS), and the even distribution of microglial cells between mutant and control BALB/cJ mice (b). (cg) The Ush1 retinal phenotype was also rescued by antioxidant (taurine) supplementation. ERG measurements on 9-month-old Ush1c−/− BALB/cJ mice treated with 0.1 M taurine (purple graphs) showed that this treatment significantly prevented the loss of retinal function. In all dark-adapted (c), photopic (d) and flicker (e) conditions, the results obtained were similar for treated mutant mice and age-matched BALB/cJ control mice (histograms: n = 5 for all conditions). Treatment also prevented a decrease in these parameters during aging, from three (mo 3) to nine months (mo 9), in Ush1g−/− mice (curves). Taurine supplementation also prevented the mislocalization of S opsin (f) and microglial cells (g) observed in Ush1c−/− mice not treated with taurine. The data shown are means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.005; (Student’s t-test). The scale bars represent 15 µm in (b) and (g), and 10 µm in (f).
Figure 7
Figure 7
Special diet prevents cone dysfunction and opsin mislocalization. (ac) In ERG measurements under dark-adapted (a and b), photopic (c) conditions at month 7, the results obtained for Ush1g−/− BALB/cJ mice fed the RM3 diet (a-wave: p = 0.1475, b-wave: p = 0.2744, photopic: p = 0.0806; see Methods) (dark red graphs) were similar to those for age-matched Wild-type (WT) controls (grey). (d) However, flicker recordings continued to be significantly lower for Ush1g−/− BALB/cJ mice (n = 6) than for wild-type control mice (n = 8) (p = 0.014). (eg) Unlike the Ush1g−/− BALB/cJ mice (f), mutant mice fed by RM3 diet (g) display no difference in retinal morphology, as illustrated by immunolabeling for S opsin that is similar to that in age-matched controls (e). *p < 0.05 (Student’s t-test). The scale bars represent 10 µm in (e, and fg).

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