Dicer Loss in Müller Glia Leads to a Defined Sequence of Pathological Events Beginning With Cone Dysfunction

Abstract

Purpose: The loss of Dicer in Müller glia (MG) results in severe photoreceptor degeneration, as it occurs in retinitis pigmentosa or age-related macular degeneration; however, the sequence of events leading to this severe degenerative state is unknown. The aim of this study was to conduct a chronological functional and structural characterization of the pathological events in MG-specific Dicer-conditional knockout (cKO) mice in vivo and histologically.

Methods: To delete Dicer and mature microRNAs (miRNAs) in MG, two conditional Dicer1 knockout mouse strains (Rlbp-CreER:tdTomato:Dicer-cKOMG and Glast-CreER:tdTomato:Dicer-cKOMG) were created. Optical coherence tomography (OCT), electroretinograms (ERGs), and histological analyses were conducted to investigate structural and functional changes up to 6 months after Dicer deletion.

Results: Dicer/miRNA loss in MG leads to (1) impairments of the area spanning from the external limiting membrane (ELM) to the retinal pigment epithelium (RPE), (2) cone photoreceptor dysfunction, and (3) retinal remodeling and functional loss of the inner retina at 1, 3, and 6 months after Dicer loss, respectively, in both of the knockout mouse strains. Furthermore, in the Rlbp-CreER:tdTomato:Dicer-cKOMG strain, rod photoreceptor impairment was found 4 months after Dicer depletion (4) accompanied by alteration of RPE integrity (5).

Conclusions: MG Dicer loss in the adult mouse retina impacts cone function prior to any measurable changes in rod function, suggesting a pivotal role for MG Dicer and miRNAs in supporting cone health. A partially impaired RPE, however, seems to accelerate rod degeneration and overall degenerative events.

Figure 1.

miRNA loss in MG results in retinal degeneration at late stage. (A) Experimental design showing Cre activation at P11 to P14 and time points of analysis 1 and 6 months after Cre induction (red stars). (B–M) Immunofluorescent labeling with antibodies against RFP (Rlbp1:Tomato), glutamine synthetase (GS), and SOX9, as well as DAPI nuclear staining of center areas of Rlbp-Cre wildtype (WT) mice (B–G) or Rlbp-Cre:Dicer-cKO_MG mice (H–M), 1 or 6 months after Cre induction. The insets in C, F, I, and L are shown in D, G, J, and M, respectively. Red arrowheads in J indicate displaced MG. Arrows in K show RPE–ELM interactions. Arrowheads in K and L show MG seal formation. Arrows in M show GS-negative cells. Scale bars: 200 µm (B, E, H, K), 50 µm (C, F, I, L), 25 µm (D, G, J, M).

Figure 2.

Early-stage MG alterations have no impact on retinal structure and function. (A) Experimental design. (B) OCT images of WT and Rlbp-Cre:Dicer-cKO_MG (R-cKO_MG, cKO) center retinas (1300-µm diameter) at the nasal–temporal axis 1 month after Cre induction. (C) Spider plots of the overall retinal thickness (diameter, µm) measured at the nasal–temporal and superior–inferior axes of WT mice (n = 4) and Rlbp-Cre:Dicer-cKO_MG mice (n = 6). (D) Spider plots of the thickness (µm) of the ELM–RPE, ONL, INL, and IPL. (E) Fullfield scotopic ERG recordings showing a-wave and b-wave amplitudes of WT mice (n = 7) and Rlbp-Cre:Dicer-cKO_MG mice (n = 6) 1 month after Cre induction. (F) Estimated saturated amplitudes (V_max, responsiveness), semi-saturation (K, sensitivity), and slope (n, heterogeneity) using the Naka–Rushton equation. OCT shown as mean ± SD; ERG shown as mean ± SEM. Significant differences are indicated. *P ≤ 0.05, Mann–Whitney U test. Scale bars: 200 µm (B). Layer explanation is given in Figure 1.

Figure 3.

Intermediate-stage retinas display structural abnormalities but no functional impairments. (A) Experimental design. (B) OCT images of WT or Rlbp-Cre:Dicer-cKO_MG mice (R-cKO_MG, cKO, center retinas, 1300-µm diameter) 3 months after Cre induction at the nasal–temporal axis. Arrowheads indicate hyperreflective foci. (C) Spider plots of the overall retinal thickness (diameter, µm) measured at the nasal–temporal and superior–inferior axes of WT mice (n = 7) and Rlbp-Cre:Dicer-cKO_MG mice (n = 11). (D) Spider plots of the thickness (µm) of the ELM–RPE, ONL, INL, and IPL. (E) Fullfield scotopic ERG recordings showing a-wave and b-wave amplitudes of WT mice (n = 10) and Rlbp-Cre:Dicer-cKO_MG mice (n = 8). (F) Estimated maximum amplitudes (V_max, responsiveness), semi-saturation (K, sensitivity), and slope (n, heterogeneity) using the Naka–Rushton equation. OCT shown as mean ± SD; ERG shown as mean ± SEM. Significant differences are indicated. *P ≤ 0.05, Mann–Whitney U test. Scale bars: 200 µm (B). Layer explanation is given in Figure 1.

Figure 4.

The miRNA-depleted MG cause photoreceptor loss and vision impairment at the late stage. (A) Experimental design. (B) OCT images of WT or Rlbp-Cre:Dicer-cKO_MG mice (R-cKO_MG, cKO, center retinas; 1300-µm diameter) 6 months after Cre induction at the nasal–temporal axis. Arrows indicate hyperreflective foci. (C) Spider plots of the overall retinal thickness (diameter, µm) measured at the nasal–temporal and superior–inferior axes of WT mice (n = 4) and Rlbp-Cre:Dicer-cKO_MG mice (n = 7). (D) Spider plots of the thickness (µm) of the ELM–RPE, ONL, INL, and IPL. (E) Fullfield scotopic ERG recordings showing a-wave and b-wave amplitudes of WT mice (n = 5) and Rlbp-Cre:Dicer-cKO_MG mice (n = 5) 6 months after Cre induction. (F) Estimated maximum amplitudes (V_max, responsiveness), semi-saturation (K, sensitivity), and slope (n, heterogeneity) using the Naka–Rushton equation. OCT shown as mean ± SD; ERG shown as mean ± SEM. Significant differences are indicated. *P ≤ 0.05, Mann–Whitney U test. Scale bars: 200 µm (B). Layer explanation is given in Figure 1.

Figure 5.

Retinal remodeling in late-stage retinas with miRNA-depleted MG. (A) Experimental design. (B–G) Immunofluorescent labeling with antibodies against RFP (Rlbp1:Tomato), calretinin (B, E), calbindin (C, F), and ChAT (D, G), as well as DAPI nuclear staining of center areas of WT mice (B–D) or Rlbp-Cre:Dicer-cKO_MG mice (E–G), 6 months after Cre induction. Arrowheads in E, F, and G show a glial seal; the yellow arrow in E indicates a neuronal cell body beneath the glial seal; the red arrow in the high-magnification inset in E shows invagination; the white arrow in F indicates RPE–ELM interaction/fusion. Scale bars: 50 µm. Layer explanation is given in Figure 1.

Figure 6.

Rod functional impairments are evident 4 months after MG miRNA depletion. (A) Experimental design. (B, E) OCT images of central and peripheral retinal areas of Rlbp-Cre:Dicer-cKO_MG mice 4 months (B) and 5 months (E) after Cre induction. Arrowheads indicate structural impairments, and arrows indicate enlarged INL. (C) Spider plots of the overall retinal thickness at the nasal–temporal and superior–inferior axes of WT mice (n = 11), 4-month mice (n = 5), and 5-month Rlbp-Cre:Dicer-cKO_MG mice (n = 5). (D) Spider plots of the thickness (µm) of the ELM–RPE, ONL, INL, and IPL. (F) Timeline plots showing the averaged thickness of Rlbp-Cre:Dicer-cKO_MG mice over time for the total retinal and specific layers. (G) Fullfield scotopic electroretinogram recordings showing a-wave and b-wave amplitudes of 4-month Rlbp-Cre:Dicer-cKO_MG mice (n = 5) in comparison to WT mice (n = 15). (H) Estimated maximum amplitudes (V_max, responsiveness) semi-saturation (K, sensitivity), and slope (n, heterogeneity) using the Naka–Rushton equation. OCT shown as mean ± SD; ERG shown as mean ± SEM. Significant differences are indicated. *P ≤ 0.05, Mann–Whitney U test. Scale bars: 200 µm (B, E). Layer explanation is given in Figure 1.

Figure 7.

Rlbp-Cre:Dicer-cKO mice display loss of cone function 3 month after Cre induction. (A) Experimental design. (B–D) Fullfield photopic ERG recordings showing representative ERG waveforms and b-wave intensity–amplitude plots for WT and Rlbp-Cre:Dicer-cKO_MG mice at 3 months (WT, n = 10; cKO, n = 8) (B), 4 months (WT, n = 15; cKO, n = 5) (C), and 6 months (WT, n = 5; cKO, n = 5) (D). (E–H’) Immunofluorescent labeling of cross-sections (E, G) and flatmounts (F/F’, H/H’) with antibodies against RFP (Rlbp1:Tomato) and M opsin (medium-wavelength cone opsin) of center areas of Rlbp-Cre WT mice (E, F) and 3-month Rlbp-Cre:Dicer-cKO_MG mice (G, H). Insets in F and H are shown in F’ and H’. Arrowheads indicate areas with reduced and scattered M opsin expression. Values are expressed as mean ± SEM. Significant differences are indicated. *P ≤ 0.05, Mann–Whitney U test. Scale bars: 50 µm (E, G, F’, H’) and 200 µm (F, H). Layer explanation is given in Figure 1.

Figure 8.

Rlbp-Cre:Dicer-cKO mice have partially impaired RPE. (A–C) Immunofluorescent labeling with antibodies against RFP (Rlbp1-Tomato), ZO-1, OTX2, and DAPI nuclear staining RPE flatmounts of 3-month Rlbp-Cre WT mice (A), 3-month Rlbp-Cre:Dicer-cKO mice (B), and 6-month Rlbp-Cre:Dicer-cKO mice (C). Insets in A to C are shown in A’ to C’. Arrows in B/B’ show altered nuclei locations in the 3-month Rlbp-Cre:Dicer-cKO mice. Arrows in C’ show enlarged RPE cells or RPE cells that lost their ZO-1⁺ membrane. Arrowheads in C’ show Rlbp-Cre:Dicer-cKO RPE cells with reduced ZO-1 and OTX2 expression. Scale bars: 50 µm (A–C) and 25 µm (A’–C’).

Figure 9.

Glast-Cre:Dicer-cKO mice display hyperreflective foci and retinal thinning. (A) Experimental design. (B) OCT images of WT and or Glast-Cre:Dicer-cKO_MG center at the nasal–temporal axis 3 months after Cre induction. (C) Immunofluorescent labeling of Glast-Cre:Dicer-cKO RPE flatmounts 3 months after Cre induction with antibodies against RFP (Glast:Tomato), ZO-1, OTX2, and DAPI. (D) OCT images of Glast-Cre:Dicer-cKO_MG center at the nasal–temporal axis 6 months after Cre induction. Arrowheads in B and D indicate structural impairments. (E) Spider plots of the overall retinal thickness at the nasal–temporal and superior–inferior axis of WT mice (n = 5) and Glast-Cre:Dicer-cKO_MG mice (n = 6) 6 months after Cre induction, in comparison to age-matched Rlbp-Cre:Dicer-cKO_MG mice (n = 7). (F) Spider plots of the thickness (µm) of the retinal layers for WT and Glast-Cre:Dicer-cKO_MG mice, in comparison to Rlbp-Cre:Dicer-cKO_MG mice. Values are expressed as mean ± SD. Significant differences are indicated. *Wildtype-cKO comparison, +cKO-cKO comparison: P ≤ 0.05, Mann–Whitney U test. Scale bars: 200 µm (B–D) and 25 µm (inset). Layer explanation is given in Figure 1.

Figure 10.

Glast-Cre:Dicer-cKO mice show loss of cone function 3 months after Dicer deletion. (A, B) Fullfield photopic ERG recordings showing representative ERG waveforms and b-wave intensity–amplitude plots of 3-month Glast-Cre:Dicer-cKO_MG mice (n = 5) in comparison to WT mice (n = 5) (A) and 6-month Glast-Cre:Dicer-cKO_MG mice (n = 6) in comparison to WT mice (n = 5) (B). (C, D) Immunofluorescent labeling of retinal cross-sections with antibodies against RFP (Glast:tdTomato) and M opsin (medium-wavelength cone opsin) of center areas of WT mice (C) and 3-month Glast-Cre:Dicer-cKO_MG mice (D). (E) Fullfield scotopic ERG recordings showing a-wave and b-wave amplitudes of Glast-Cre:Dicer-cKO_MG (n = 6) in comparison to WT (n = 5) and Rlbp-Cre:Dicer-cKO_MG (n = 5). (F, F’) Immunofluorescent labeling of central retinal areas of the Glast-Cre:Dicer-cKO_MG mouse 6 months after Dicer deletion, with antibodies against RFP (Glast:Tomato), GS, and SOX9, as well as DAPI nuclear staining. Arrows in F show a disturbed ELM, arrowheads in F’ display GS-reduced MG. Scale bars: 50 µm (C, D, F) and 25 µm (F’). Values are expressed as mean ± SEM. Significant differences are indicated. *WT-cKO comparison, +cKO-cKO comparison: P ≤ 0.05, Mann–Whitney U test. Layer explanation is given in Figure 1.

Figure 11.

Dicer-depleted MG lead to cone functional loss with subsequent rod loss. Schematic overview of the major events happening in the MG/RPE-driven retinal degeneration model (Rlbp-CreER:Dicer-cKO) and the MG-driven retinal degeneration model (Glast-CreER:Dicer-cKO) emphasizing the shared pathological features: cone outer segment impairment and cone functional loss and hyperreflective foci in the ELM–RPE at the intermediate state and rod degeneration/rod functional loss and interneuron degeneration leading to retinal remodeling at the late stage. Additional features evident in both models include ELM breakage and glial seal formation. The Rlbp-CreER:Dicer-cKO model displayed furthermore impaired RPE contributing to rod loss and accelerating the overall pathological phenotype.